Beta-lactamases with improved properties for therapy

ABSTRACT

This invention relates to, in part, compositions of beta-lactamases and methods of using these enzymes in, for example, gastrointestinal tract (GI tract) disorders such as C. difficile infection (CDI).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/993,159, filed on May 30, 2018 (now U.S. Pat. No. 10,087,433), whichis a continuation of U.S. patent application Ser. No. 15/674,177, filedon Aug. 10, 2017 (now U.S. Pat. No. 10,011,824), which is a continuationof U.S. patent application Ser. No. 15/611,881, filed Jun. 2, 2017 (nowU.S. Pat. No. 9,783,797), which is a continuation of U.S. patentapplication Ser. No. 15/245,517, filed Aug. 24, 2016 (now U.S. Pat. No.9,695,409), which is a continuation of U.S. patent application Ser. No.15/200,508, filed Jul. 1, 2016 (now U.S. Pat. No. 9,464,280), which is acontinuation of U.S. patent application Ser. No. 15/160,669 (now U.S.Pat. No. 9,404,103), filed May 20, 2016, which is a continuation of U.S.patent application Ser. No. 15/019,474, filed Feb. 9, 2016 (now U.S.Pat. No. 9,376,673), which is a continuation of U.S. patent applicationSer. No. 14/689,877, filed Apr. 17, 2015 (now U.S. Pat. No. 9,290,754),which claims the benefit of U.S. Provisional Patent Application No.61/980,844, filed Apr. 17, 2014, and U.S. Provisional Patent ApplicationNo. 62/046,627, filed Sep. 5, 2014, all of which are hereby incorporatedby reference in their entirety.

FIELD OF THE INVENTION

This invention relates to, in part, compositions of beta-lactamases andmethods for using these enzymes in, for example, gastrointestinal tract(GI tract) disorders such as C. difficile infection (CDI).

BACKGROUND

Humans may be considered to be a “superorganism” which is a conglomerateof mammalian and microbial cells, with the latter estimated to outnumberthe former by ten to one. This microbial component, and its microbialgenetic repertoire, the microbiome, is roughly 100-times greater thanthat of the human host. Strikingly, despite this enormous diversity offoreign organisms, the human immune system generally maintains a stateof synergy. This is particularly true of the distal GI tract, whichhouses up to 1000 distinct bacterial species and an estimated excess of1×10¹⁴ microorganisms, and appears to be central in defining human hosthealth status. Loss of the careful balance in the microbiome, especiallyin the GI tract, can lead to various diseases.

However, antibiotic medical treatments, which are needed to treatcertain aspects of disease, can induce disruption in the microbiome,including in the GI tract, and lead to further disease. For instance,certain parentally administered beta-lactams like ampicillin,ceftriaxone, cefoperazone, and piperacillin are, in part, eliminated viabiliary excretion into the proximal part of the small intestine(duodenum). Residual unabsorbed beta-lactams in the intestinal tract maycause an undesirable effect on the ecological balance of normalintestinal microbiota resulting in, for example, CDI,antibiotic-associated diarrhea, overgrowth of pathogenic bacteria suchas vancomycin resistant enterococci (VRE), extended-spectrumbeta-lactamase producing Gram-negative bacilli (ESBL), and fungi, andselection of antibiotic-resistance strains among both normal intestinalmicrobiota and potential pathogen bacteria.

One approach for avoiding or rebalancing the ecological balance ofnormal intestinal microbiota is the therapeutic use of beta-lactamases,for example, by inactivating excreted or unabsorbed antibiotics in theGI tract, thereby maintaining a normal intestinal microbiota andpreventing its overgrowth with potentially pathogenic micro-organisms.

There remains a need for agents and medicaments for the treatment ofgastrointestinal tract (GI tract) disorders such as C. difficileinfection (CDI) which have enzymatic properties that are best suited forthese uses.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides for beta-lactamasescomprising an amino acid sequence that is based on the P1Abeta-lactamase but containing advantageous mutations that, for example,change substrate specificity and/or activity and provide for therapeuticadvantages. For example, such mutants can have at least 70% sequenceidentity with SEQ ID NO: 1 and have one or more mutations at thefollowing Ambler classification positions F33, Q135, G156, A232, A237,A238, S240, T243, R244, S266, and D276. Such beta-lactamases have theability to hydrolyze both penicillins and cephalosporins and do so withenzymatically desirable characteristics, such as low K_(M)s and/or highV_(max)s for select antibiotic substrates.

These improved beta-lactamases find uses in a number of therapies,including the prevention or treatment of CDI and/or a C.difficile-associated disease or other antibiotic-induced adverse effectsin the GI tract. For example, the beta-lactamases find use in allowing apatient to undergo antibiotic therapy while being protected againstdisease that could result from excess antibiotics negatively affectingthe microbiome. Such use does not interfere with the systemic utility ofthe antibiotic. Rather, the beta-lactamases clear out excess antibioticthat may populate parts of the GI tract and, in doing so, prevent thedisruption of the microbiota that is linked to the various diseasestates described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a 12% Bis-Tris Criterion XT SDS-PAGE (BioRad) gel withconcentrated samples (see labels of the samples to the right of the gel)from a 10 g/l glucose B. subtilis growth. MWM=molecular weight markerPrecision Plus (BioRad), RS310=P1A B. subtilis strain and P1A A18K31=P1Areference material. “P1A→” denotes the right size of P1A and the mutantproteins.

FIG. 2 shows a 12% Bis-Tris Criterion XT SDS-PAGE (BioRad) gel withsupernatant samples (see labels of the samples to the right of the gel)from a 5 g/l glucose B. subtilis growth. 14 μl of each supernatant wasapplied on the gel. MWM=molecular weight marker Precision Plus (BioRad),RS310=P1A B. subtilis strain and “P1A→” denotes the right size of P1Aand the mutant proteins.

FIG. 3 shows an elution diagram from a HIC run. The places of collectedelution fractions are marked with arrows and black dashed lines (dashedlines farthest to the right) both perpendicular to the X-axis.Flow-through fractions A7-B8 can be easily seen on the diagram. The A₂₈₀graph is represented with a solid line (top line), conductivity of theeluted solution by a solid line (second line from the top). Sampleinjection is marked with a dashed line (dashed line farthest to theleft) and fractions with multiple solid lines both perpendicular to theX-axis.

FIG. 4 shows an elution diagram from a HIC run. The places of collectedelution fractions are marked with arrows and black dashed lines (dashedlines farthest to the right) both perpendicular to the X-axis.Flow-through fractions A1-A8 can be easily seen on the diagram. The A280graph is represented with a solid line (second line from the top),conductivity of the eluted solution by a solid line (top line). Sampleinjection is marked with a dashed line (dashed line farthest to theleft) and fractions with multiple solid lines both perpendicular to theX-axis.

FIG. 5 shows an elution diagram from a HIC run. The places of collectedelution fractions are marked with arrows and black dashed lines (dashedlines farthest to the right) both perpendicular to the X-axis.Flow-through fractions A1-B1 can be easily seen on the diagram. The A₂₈₀graph is represented with a solid line (second line from the top),conductivity of the eluted solution by a solid line (top line). Sampleinjection is marked with a dashed line (dashed line farthest to theleft) and fractions with multiple solid lines both perpendicular to theX-axis. The section before zero at the X-axis represents the columnequilibration with buffer A.

FIG. 6 shows a SDS-PAGE gel of the fractions of the three HICpurifications shown in FIGS. 3-5. AS-sup=ammonium sulphate filtrate usedin the particular purification, i.e. starting material for purification.B1 161008, B7 161008, B8 161008, A2 041108, A7 041108, and B1 121108:flow through fractions of each of purifications. C3-C8 161008, C9-D1161008, D2-D8 161008, B2-B10 041108, and B8-C3 121108: eluted proteinpeak, pooled fractions. A18K31 P1A: reference material, 0.64 μg/lane.

FIG. 7 demonstrates the SOE (spliced overlap extension) technique. Thetarget amino acids of the B3 β-strand of P1A are so closely situated toeach other that all four amino acids can be exchanged using the SOEtechnique. The region encompassing the exchanges contains 24 nucleotides(8 amino acid codons) and is included in the overlap extension part oftwo PCR primers. Mutated penP gene is first amplified by PCR in twoparts, using primers A+B and C+D. The two PCR products are then combinedand amplified using primers A and D, complementary to the 5′ and 3′parts of the penP gene. Cloning sites are included in primers A and D.Once the construct is ready, further mutations can be added using theMulti kit. The amino acid sequences encoded by the overlapping primers(B and C) are shown.

FIG. 8 demonstate antibiotic degradation activity of P1A (left bars) andP4A (right bars). Concentrations of 10 or 100 ng/ml of enzyme are usedagainst the indicated antibiotics. The data are plotted as the highestantibiotic concentration that is degraded by the indicatedbeta-lactamase (the antibiotic concentrations used were 10, 100, or 1000μg/ml).

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of certainmutant beta-lactamase enzymes that have desirable characteristics foruse in therapy, including, for example, an ability to hydrolyze one ormore of penicillins and cephalosporins.

In some aspects, the present invention provides for beta-lactamases, andpharmaceutical compositions of these beta-lactamases. In variousaspects, the beta-lactamases and/or pharmaceutical compositions arederived by mutation from SEQ ID NO: 1 (Bacillus licheniformis PenP, i.e.P1A). Accordingly, in various aspects, the present invention providesfor beta-lactamases, and/or pharmaceutical compositions which are P1Aderivatives.

P1A was designed to inactivate parenterally administered penicillingroup beta-lactams (e.g. penicillin, amoxicillin ampicillin andpiperacillin) with or without beta-lactamase inhibitors (e.g.tazobactam, sulbactam, clavulanic acid) excreted via biliary system (seeInternational Patent Publications WO 1993/013795 and WO 2008/065247;Tarkkanen, A. M. et al., Antimicrob Agents Chemother. 53: 2455, thecontents of which are hereby incorporated by reference in theirentireties). The P1A enzyme is a recombinant form of Bacilluslicheniformis 749/C small exo beta-lactamase (WO 2008/065247) whichbelongs to class A and is grouped to subgroup 2a in functionalclassification. B. licheniformis beta-lactamase and its P1A derivativeare considered as penicillinases which have high hydrolytic capacity todegrade e.g. penicillin, ampicillin, amoxicillin or piperacillin andthey are generally inhibited by active site-directed beta-lactamaseinhibitors such as clavulanic acid, sulbactam or tazobactam. However,the P1A enzyme has a limited capacity to inactivate beta-lactamantibiotics that belong to the cephalosporin or the carbapenem group.Because the employed beta-lactamases possess poor activity tocephalosporins, they cannot be applied in conjunction with parenteralcephalosporin therapy for inactivation of unabsorbed beta-lactam in thesmall intestinal tract. Therefore, in some embodiments, thebeta-lactamases and/or pharmaceutical compositions of the presentinvention improve upon the properties of P1A, as described herein.

In various embodiments, the inventive beta-lactamases possess desirablecharacteristics, including, for example, having an ability toefficiently target a broad spectra of antibiotics. In variousembodiments, the inventive beta-lactamases possess desirable enzymekinetic characteristics. For example, in some embodiments, thebeta-lactamases possess a low K_(M) for at least one cephalosporin,including, for example, a K_(M) of less than about 500 μM, or about 100μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100 nM), or about0.01 μM (10 nM), or about 1 nM. For example, in some embodiments, thebeta-lactamases possess a low K_(M) for at least one penicillin,including, for example, a K_(M) of less than about 500 μM, or about 100μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100 nM), or about0.01 μM (10 nM), or about 1 nM. In various embodiments, the inventivebeta-lactamases possess a high V_(max) for at least one cephalosporin,including, for example, V_(max) which is greater than about 100 s-1, orabout 1000 s-1, or about 10000 s-1, or about 100000 s-1, or about1000000 s-1. In various embodiments, the inventive beta-lactamasespossess a high V_(max) for at least one penicillin, including, forexample, V_(max) which is greater than about 100 s-1, or about 1000 s-1,or about 10000 s-1, or about 100000 s-1, or about 1000000 s-1. Invarious embodiments, the inventive beta-lactamases possess catalyticefficiency is greater than about 10⁶ M⁻¹ s⁻¹ for at least onecephalosporin. In various embodiments, the inventive beta-lactamasespossess catalytic efficiency is greater than about 10⁶ M⁻¹ s⁻¹ for atleast one penicillin. In various embodiments, the inventivebeta-lactamases possess the desirable enzyme kinetic characteristics forat least one of either or both of cephalosporins and penicillins. Invarious embodiments, specific antibiotics (e.g. cephalosporins andpenicillins are described herein).

Provided herein is the 263 amino acid sequence of the P1A enzyme (afterremoval of a 31 amino acid signal sequence and the QASKT(Gln-Ala-Ser-Lys-Thr) pentapeptide at the N terminus, see SEQ ID NO: 3).As described herein, mutations may be made to this sequence to generatethe inventive beta-lactamases.

SEQ ID NO: 1 Glu Met Lys Asp Asp Phe Ala Lys Leu Glu Glu GlnPhe Asp Ala Lys Leu Gly Ile Phe Ala Leu AspThr Gly Thr Asn Arg Thr Val Ala Tyr Arg Pro AspGlu Arg Phe Ala Phe Ala Ser Thr Ile Lys Ala LeuThr Val Gly Val Leu Leu Gln Gln Lys Ser Ile GluAsp Leu Asn Gln Arg Ile Thr Tyr Thr Arg Asp AspLeu Val Asn Tyr Asn Pro Ile Thr Glu Lys His ValAsp Thr Gly Met Thr Leu Lys Glu Leu Ala Asp AlaSer Leu Arg Tyr Ser Asp Asn Ala Ala Gln Asn LeuIle Leu Lys Gln Ile Gly Gly Pro Glu Ser Leu LysLys Glu Leu Arg Lys Ile Gly Asp Glu Val Thr AsnPro Glu Arg Phe Glu Pro Glu Leu Asn Glu ValAsn Pro Gly Glu Thr Gln Asp Thr Ser Thr Ala ArgAla Leu Val Thr Ser Leu Arg Ala Phe Ala Leu GluAsp Lys Leu Pro Ser Glu Lys Arg Glu Leu Leu IleAsp Trp Met Lys Arg Asn Thr Thr Gly Asp AlaLeu Ile Arg Ala Gly Val Pro Asp Gly Trp Glu ValAla Asp Lys Thr Gly Ala Ala Ser Tyr Gly Thr ArgAsn Asp Ile Ala Ile Ile Trp Pro Pro Lys Gly AspPro Val Val Leu Ala Val Leu Ser Ser Arg Asp LysLys Asp Ala Lys Tyr Asp Asp Lys Leu Ile Ala GluAla Thr Lys Val Val Met Lys Ala Leu Asn Met Asn Gly Lys.

In some embodiments, SEQ ID NO: 1 may have a Met and/or Thr preceedingthe first residue of the sequence. In various embodiments, the Met maybe cleaved. As described herein, mutations may be made to the sequencecomprising the Met and/or Thr preceeding the first residue to generatethe inventive beta-lactamases.

Also provided herein is the 299 amino acid sequence of the P1A enzymebefore removal of a 31 amino acid signal sequence and the QASKT(Gln-Ala-Ser-Lys-Thr) pentapeptide at the N terminus as SEQ ID NO: 3:

SEQ ID NO: 3 Met Ile Gln Lys Arg Lys Arg Thr Val Ser Phe ArgLeu Val Leu Met Cys Thr Leu Leu Phe Val SerLeu Pro Ile Thr Lys Thr Ser Ala Gln Ala Ser LysThr Glu Met Lys Asp Asp Phe Ala Lys Leu Glu GluGln Phe Asp Ala Lys Leu Gly Ile Phe Ala Leu AspThr Gly Thr Asn Arg Thr Val Ala Tyr Arg Pro AspGlu Arg Phe Ala Phe Ala Ser Thr Ile Lys Ala LeuThr Val Gly Val Leu Leu Gln Gln Lys Ser Ile GluAsp Leu Asn Gln Arg Ile Thr Tyr Thr Arg Asp AspLeu Val Asn Tyr Asn Pro Ile Thr Glu Lys His ValAsp Thr Gly Met Thr Leu Lys Glu Leu Ala Asp AlaSer Leu Arg Tyr Ser Asp Asn Ala Ala Gln AsnLeu Ile Leu Lys Gln Ile Gly Gly Pro Glu Ser LeuLys Lys Glu Leu Arg Lys Ile Gly Asp Glu Val ThrAsn Pro Glu Arg Phe Glu Pro Glu Leu Asn Glu ValAsn Pro Gly Glu Thr Gln Asp Thr Ser Thr AlaArg Ala Leu Val Thr Ser Leu Arg Ala Phe Ala LeuGlu Asp Lys Leu Pro Ser Glu Lys Arg Glu LeuLeu Ile Asp Trp Met Lys Arg Asn Thr Thr Gly AspAla Leu Ile Arg Ala Gly Val Pro Asp Gly Trp GluVal Ala Asp Lys Thr Gly Ala Ala Ser Tyr Gly ThrArg Asn Asp Ile Ala Ile Ile Trp Pro Pro Lys Gly AspPro Val Val Leu Ala Val Leu Ser Ser Arg Asp LysLys Asp Ala Lys Tyr Asp Asp Lys Leu Ile Ala GluAla Thr Lys Val Val Met Lys Ala Leu Asn Met Asn Gly Lys

Further, the present invention also provides for additional upstreamresidues from the first residue of SEQ ID NO: 1 (see, e.g., JBC 258(18): 11211, 1983, the contents of which are hereby incorporated byreference—including the exo-large and exo-small versions of penP andpenP1). Further, the present invention also provides for additionaldownstream residues from the last residue of SEQ ID NO: 1.

The polynucleotide sequence of P1A (after removal of a 31 amino acidsignal sequence and the QAKST pentapeptide at the N terminus) is alsoprovided as SEQ ID NO: 2. As described herein, mutations may be made tothis sequence to generate the inventive beta-lactamases (including,taking into account degeneracy of the genetic code).

SEQ ID NO: 2 gagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

Also provided is the polynucleotide sequence of P1A before the removalof a 31 amino acid signal sequence and the QASKT pentapeptide at the Nterminus as SEQ ID NO: 4. As described herein, mutations may be made tothis sequence to generate the inventive beta-lactamases (including,taking into account degeneracy of the genetic code).

SEQ ID NO: 4 atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagcgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtggctgataaaactggagcggcatcatatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgatgataaacttattgcagaggcaacaaaggtggtaatgaaagccttaaacatgaacggcaaataa

In some embodiments, the invention pertains to mutagenesis of abeta-lactamase (e.g. a class A beta-lactamase) to derive advantageousenzymes (e.g. those that can target a broad spectra of antibiotics). Insome embodiments, the invention includes site-directed mutagenesis,random mutagenesis, and/or directed evolution approaches. In someembodiments, mutation design is based on, inter alia, structural data(e.g. crystal structure data, homolog models, etc.) of the following:P1A crystal structure (Knox and Moews, J. Mol Biol., 220, 435-455(1991)), CTX-M-44 (1BZA (Ibuka et al. Journal of Molecular BiologyVolume 285, Issue 5 2079-2087 (1999), 1IYS (Ibuka et al. Biochemistry,2003, 42 (36): 10634-43), 1IYO, 1IYP and 1IYQ (Shimamura et al 2002 J.Biol. Chem. 277:46601-08), Proteus vulgaris K1 (1HZO, Nugaka et al. JMol Biol. 2002 Mar. 15; 317(1):109-17) and Proteus penneri HugA(Liassine et al. Antimicrob Agents Chemother. 2002 January; 46(1):216-9.2002), and reviewed in Bonnet, Antimicrob. Agents Chemother 48(1): 1-14(2004) (for CTM-X), the contents of all of these documents are herebyincorporated by reference in their entirety). In some embodiments, thepresent mutations are informed by analysis of structural data (e.g.crystal structure data, homolog models, etc.) of any one of thefollowing beta-lactamases: P1A (see, e.g. U.S. Pat. No. 5,607,671, thecontents of which are hereby incorporated by reference), P2A (see, e.g.,WO 2007/147945, the contents of which are hereby incorporated byreference), P3A (see, e.g., WO 2011/148041, the contents of which arehereby incorporated by reference), CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-9,CTX-M-10, CTX-M-14, CTX-M-15, CTX-M-16, CTX-M-18, CTX-M-19, CTX-M-25,CTX-M-26, CTX-M-27, CTX-M-32, CTX-M-44, CTX-M-45, and CTX-M-54. Suchinformation is available to one skilled in the art at known databases,for example, Swiss-Prot Protein Sequence Data Bank, NCBI, and PDB.

In some embodiments, the invention pertains to one or more (e.g. about1, or about 2, or about 3, or about 4, or about 5, or about 6, or about7, or about 8, or about 9, or about 10, or about 15, or about 20, orabout 30, or about 40, or about 50, or about 60, or about 70, or about80, or about 90, or about 100, or about 110, or about 120, or about 130,or about 140, or about 150) mutations to SEQ ID NO: 1 or SEQ ID NO: 3 ora sequence with at least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5, 99.8, 99.9% identity to SEQ ID NO: 1 or SEQ IDNO: 3 (or about 70%, or about 75%, or about 80%, or about 85%, or about90, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%identity to SEQ ID NO: 1 or SEQ ID NO: 3). In various embodiments, oneor more amino acid of SEQ ID NO: 1 or SEQ ID NO: 3 is substituted with anaturally occurring amino acid, such as a hydrophilic amino acid (e.g. apolar and positively charged hydrophilic amino acid, such as arginine(R) or lysine (K); a polar and neutral of charge hydrophilic amino acid,such as asparagine (N), glutamine (Q), serine (S), threonine (T),proline (P), and cysteine (C), a polar and negatively chargedhydrophilic amino acid, such as aspartate (D) or glutamate (E), or anaromatic, polar and positively charged hydrophilic amino acid, such ashistidine (H)) or a hydrophobic amino acid (e.g. a hydrophobic,aliphatic amino acid such as glycine (G), alanine (A), leucine (L),isoleucine (I), methionine (M), or valine (V), a hydrophobic, aromaticamino acid, such as phenylalanine (F), tryptophan (W), or tyrosine (Y)or a non-classical amino acid (e.g. selenocysteine, pyrrolysine,N-formylmethionine β-alanine, GABA and δ-Aminolevulinic acid.4-Aminobenzoic acid (PABA), D-isomers of the common amino acids,2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, ε-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosme, citrulline, homocitrulline, cysteicacid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β methylamino acids, C α-methyl amino acids, N α-methyl amino acids, and aminoacid analogs in general).

In illustrative embodiments, inventive mutations include, but are notlimited to one or more (e.g. about 1, or about 2, or about 3, or about4, or about 5, or about 6, or about 7, or about 8, or about 9, or about10, or about 15, or about 20, or about 30, or about 40, or about 50, orabout 60, or about 70, or about 80, or about 90, or about 100, or about110, or about 120, or about 130, or about 140, or about 150) of thefollowing mutations to SEQ ID NO: 1 or SEQ ID NO: 3 or a sequence withat least 30, 35, 40, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 99.5, 99.8, 99.9% identity to SEQ ID NO: 1 or SEQ ID NO: 3 (or about70%, or about 75%, or about 80%, or about 85%, or about 90, or about95%, or about 96%, or about 97%, or about 98%, or about 99% identity toSEQ ID NO: 1 or SEQ ID NO: 3): Glu1Ala; Glu1Cys; Glu1Asp; Glu1Phe;Glu1Gly; Glu1His; Glu1 Ile; Met1Lys; Glu1Leu; Glu1Met; Glu1Asn; Glu1Pro;Glu1Gln; Glu1Arg; Glu1Ser; Glu1Thr; Glu1Val; Glu1Trp; Glu1Tyr; Met2Ala;Met2Cys; Met2Asp; Met2Glu; Met2Phe; Met2Gly; Met2His; Met2Ile; Met1Lys;Met2Leu; Met2Asn; Met2Pro; Met2Gln; Met2Arg; Met2Ser; Met2Thr; Met2Val;Met2Trp; Met2Tyr; Lys3Ala; Lys3Cys; Lys3Asp; Lys3Glu; Lys3Phe; Lys3Gly;Lys3His; Lys3Ile; Lys3Leu; Lys3Met; Lys3Asn; Lys3Pro; Lys3Gln; Lys3Arg;Lys3Ser; Lys3Thr; Lys3Val; Lys3Trp; Lys3Tyr; Asp4Ala; Asp4Cys; Asp4Glu;Asp4Phe; Asp4Gly; Asp4His; Asp4Ile; Asp4Lys; Asp4Leu; Asp4Met; Asp4Asn;Asp4Pro; Asp4Gln; Asp4Arg; Asp4Ser; Asp4Thr; Asp4Val; Asp4Trp; Asp4Tyr;Asp5Ala; Asp5Cys; Asp5Glu; Asp5Phe; Asp5Gly; Asp5His; Asp5Ile; Asp5Lys;Asp5Leu; Asp5Met; Asp5Asn; Asp5Pro; Asp5Gln; Asp5Arg; Asp5Ser; Asp5Thr;Asp5Val; Asp5Trp; Asp5Tyr; Phe6Ala; Phe6Cys; Phe6Asp; Phe6Glu; Phe6Gly;Phe6His; Phe6Ile; Phe6Lys; Phe6Leu; Phe6Met; Phe6Asn; Phe6Pro; Phe6Gln;Phe6Arg; Phe6Ser; Phe6Thr; Phe6Val; Phe6Trp; Phe6Tyr; Ala7Cys; Ala7Asp;Ala7Glu; Ala7Phe; Ala7Gly; Ala7His; Ala7Ile; Ala7Lys; Ala7Leu; Ala7Met;Ala7Asn; Ala7Pro; Ala7Gln; Ala7Arg; Ala7Ser; Ala7Thr; Ala7Val; Ala7Trp;Ala7Tyr; Lys8Ala; Lys8Cys; Lys8Asp; Lys8Glu; Lys8Phe; Lys8Gly; Lys8His;Lys8Ile; Lys8Leu; Lys8Met; Lys8Asn; Lys8Pro; Lys8Gln; Lys8Arg; Lys8Ser;Lys8Thr; Lys8Val; Lys8Trp; Lys8Tyr; Leu9Ala; Leu9Cys; Leu9Asp; Leu9Glu;Leu9Phe; Leu9Gly; Leu9His; Leu9Ile; Leu9Lys; Leu9Met; Leu9Asn; Leu9Pro;Leu9Gln; Leu9Arg; Leu9Ser; Leu9Thr; Leu9Val; Leu9Trp; Leu9Tyr; Glu10Ala;Glu10Cys; Glu10Asp; Glu10Phe; Glu10Gly; Glu10His; Glu10Ile; Glu10Lys;Glu10Leu; Glu10Met; Glu10Asn; Glu10Pro; Glu10Gln; Glu10Arg; Glu10Ser;Glu10Thr; Glu10Val; Glu10Trp; Glu10Tyr; Glu11Ala; Glu11Cys; Glu11Asp;Glu11Phe; Glu11Gly; Glu11His; Glu11Ile; Glu11Lys; Glu11Leu; Glu11Met;Glu11Asn; Glu11Pro; Glu11Gln; Glu11Arg; Glu11Ser; Glu11Thr; Glu11Val;Glu11Trp; Glu11Tyr; Gln12Ala; Gln12Cys; Gln12Asp; Gln12Glu; Gln12Phe;Gln12Gly; Gln12His; Gln12Ile; Gln12Lys; Gln12Leu; Gln12Met; Gln12Asn;Gln12Pro; Gln12Arg; Gln12Ser; Gln12Thr; Gln12Val; Gln12Trp; Gln12Tyr;Phe13Ala; Phe13Cys; Phe13Asp; Phe13Glu; Phe13Gly; Phe13His; Phe13Ile;Phe13Lys; Phe13Leu; Phe13Met; Phe13Asn; Phe13Pro; Phe13Gln; Phe13Arg;Phe13Ser; Phe13Thr; Phe13Val; Phe13Trp; Phe13Tyr; Asp14Ala; Asp14Cys;Asp14Glu; Asp14Phe; Asp14Gly; Asp14His; Asp14Ile; Asp14Lys; Asp14Leu;Asp14Met; Asp14Asn; Asp14Pro; Asp14Gln; Asp14Arg; Asp14Ser; Asp14Thr;Asp14Val; Asp14Trp; Asp14Tyr; Ala15Cys; Ala15Asp; Ala15Glu; Ala15Phe;Ala15Gly; Ala15His; Ala15Ile; Ala15Lys; Ala15Leu; Ala15Met; Ala15Asn;Ala15Pro; Ala15Gln; Ala15Arg; Ala15Ser; Ala15Thr; Ala15Val; Ala15Trp;Ala15Tyr; Lys16Ala; Lys16Cys; Lys16Asp; Lys16Glu; Lys16Phe; Lys16Gly;Lys16His; Lys16Ile; Lys16Leu; Lys16Met; Lys16Asn; Lys16Pro; Lys16Gln;Lys16Arg; Lys16Ser; Lys16Thr; Lys16Val; Lys16Trp; Lys16Tyr; Leu17Ala;Leu17Cys; Leu17Asp; Leu17Glu; Leu17Phe; Leu17Gly; Leu17His; Leu17Ile;Leu17Lys; Leu17Met; Leu17Asn; Leu17Pro; Leu17Gln; Leu17Arg; Leu17Ser;Leu17Thr; Leu17Val; Leu17Trp; Leu17Tyr; Gly18Ala; Gly18Cys; Gly18Asp;Gly18Glu; Gly18Phe; Gly18His; Gly18Ile; Gly18Lys; Gly18Leu; Gly18Met;Gly18Asn; Gly18Pro; Gly18Gln; Gly18Arg; Gly18Ser; Gly18Thr; Gly18Val;Gly18Trp; Gly18Tyr; Ile19Ala; Ile19Cys; Ile19Asp; Ile19Glu; Ile19Phe;Ile19Gly; Ile19His; Ile19Lys; Ile19Leu; Ile19Met; Ile19Asn; Ile19Pro;Ile19Gln; Ile19Arg; Ile19Ser; Ile19Thr; Ile19Val; Ile19Trp; Ile19Tyr;Phe20Ala; Phe20Cys; Phe20Asp; Phe20Glu; Phe20Gly; Phe20His; Phe20Ile;Phe20Lys; Phe20Leu; Phe20Met; Phe20Asn; Phe20Pro; Phe20Gln; Phe20Arg;Phe20Ser; Phe20Thr; Phe20Val; Phe20Trp; Phe20Tyr; Ala21Cys; Ala21Asp;Ala21Glu; Ala21Phe; Ala21Gly; Ala21His; Ala21Ile; Ala21Lys; Ala21Leu;Ala21Met; Ala21Asn; Ala21Pro; Ala21Gln; Ala21Arg; Ala21Ser; Ala21Thr;Ala21Val; Ala21Trp; Ala21Tyr; Leu22Ala; Leu22Cys; Leu22Asp; Leu22Glu;Leu22Phe; Leu22Gly; Leu22His; Leu22Ile; Leu22Lys; Leu22Met; Leu22Asn;Leu22Pro; Leu22Gln; Leu22Arg; Leu22Ser; Leu22Thr; Leu22Val; Leu22Trp;Leu22Tyr; Asp23Ala; Asp23Cys; Asp23Glu; Asp23Phe; Asp23Gly; Asp23His;Asp23Ile; Asp23Lys; Asp23Leu; Asp23Met; Asp23Asn; Asp23Pro; Asp23Gln;Asp23Arg; Asp23Ser; Asp23Thr; Asp23Val; Asp23Trp; Asp23Tyr; Thr24Ala;Thr24Cys; Thr24Asp; Thr24Glu; Thr24Phe; Thr24Gly; Thr24His; Thr24Ile;Thr24Lys; Thr24Leu; Thr24Met; Thr24Asn; Thr24Pro; Thr24Gln; Thr24Arg;Thr24Ser; Thr24Val; Thr24Trp; Thr24Tyr; Gly25Ala; Gly25Cys; Gly25Asp;Gly25Glu; Gly25Phe; Gly25His; Gly25Ile; Gly25Lys; Gly25Leu; Gly25Met;Gly25Asn; Gly25Pro; Gly25Gln; Gly25Arg; Gly25Ser; Gly25Thr; Gly25Val;Gly25Trp; Gly25Tyr; Thr26Ala; Thr26Cys; Thr26Asp; Thr26Glu; Thr26Phe;Thr26Gly; Thr26His; Thr26Ile; Thr26Lys; Thr26Leu; Thr26Met; Thr26Asn;Thr26Pro; Thr26Gln; Thr26Arg; Thr26Ser; Thr26Val; Thr26Trp; Thr26Tyr;Asn27Ala; Asn27Cys; Asn27Asp; Asn27Glu; Asn27Phe; Asn27Gly; Asn27His;Asn27Ile; Asn27Lys; Asn27Leu; Asn27Met; Asn27Pro; Asn27Gln; Asn27Arg;Asn27Ser; Asn27Thr; Asn27Val; Asn27Trp; Asn27Tyr; Arg28Ala; Arg28Cys;Arg28Asp; Arg28Glu; Arg28Phe; Arg28Gly; Arg28His; Arg28Ile; Arg28Lys;Arg28Leu; Arg28Met; Arg28Asn; Arg28Pro; Arg28Gln; Arg28Ser; Arg28Thr;Arg28Val; Arg28Trp; Arg28Tyr; Thr29Ala; Thr29Cys; Thr29Asp; Thr29Glu;Thr29Phe; Thr29Gly; Thr29His; Thr29Ile; Thr29Lys; Thr29Leu; Thr29Met;Thr29Asn; Thr29Pro; Thr29Gln; Thr29Arg; Thr29Ser; Thr29Val; Thr29Trp;Thr29Tyr; Val30Ala; Val30Cys; Val30Asp; Val30Glu; Val30Phe; Val30Gly;Val30His; Val30Ile; Val30Lys; Val30Leu; Val30Met; Val30Asn; Val30Pro;Val30Gln; Val30Arg; Val30Ser; Val30Thr; Val30Trp; Val30Tyr; Ala31Ala;Ala31Cys; Ala31Asp; Ala31Glu; Ala31Phe; Ala31Gly; Ala31His; Ala31Ile;Ala31Lys; Ala31Leu; Ala31Met; Ala31Asn; Ala31Pro; Ala31Gln; Ala31Arg;Ala31Ser; Ala31Thr; Ala31Val; Ala31Trp; Ala31Tyr; Tyr32Ala; Tyr32Cys;Tyr32Asp; Tyr32Glu; Tyr32Phe; Tyr32Gly; Tyr32His; Tyr32Ile; Tyr32Lys;Tyr32Leu; Tyr32Met; Tyr32Asn; Tyr32Pro; Tyr32Gln; Tyr32Arg; Tyr32Ser;Tyr32Thr; Tyr32Val; Tyr32Trp; Arg33Ala; Arg33Cys; Arg33Asp; Arg33Glu;Arg33Phe; Arg33Gly; Arg33His; Arg33Ile; Arg33Lys; Arg33Leu; Arg33Met;Arg33Asn; Arg33Pro; Arg33Gln; Arg33Ser; Arg33Thr; Arg33Val; Arg33Trp;Arg33Tyr; Pro34Ala; Pro34Cys; Pro34Asp; Pro34Glu; Pro34Phe; Pro34Gly;Pro34His; Pro34Ile; Pro34Lys; Pro34Leu; Pro34Met; Pro34Asn; Pro34Gln;Pro34Arg; Pro34Ser; Pro34Thr; Pro34Val; Pro34Trp; Pro34Tyr; Asp35Ala;Asp35Cys; Asp35Glu; Asp35Phe; Asp35Gly; Asp35His; Asp35Ile; Asp35Lys;Asp35Leu; Asp35Met; Asp35Asn; Asp35Pro; Asp35Gln; Asp35Arg; Asp35Ser;Asp35Thr; Asp35Val; Asp35Trp; Asp35Tyr; Glu36Ala; Glu36Cys; Glu36Asp;Glu36Phe; Glu36Gly; Glu36His; Glu36Ile; Glu36Lys; Glu36Leu; Glu36Met;Glu36Asn; Glu36Pro; Glu36Gln; Glu36Arg; Glu36Ser; Glu36Thr; Glu36Val;Glu36Trp; Glu36Tyr; Arg37Ala; Arg37Cys; Arg37Asp; Arg37Glu; Arg37Phe;Arg37Gly; Arg37His; Arg37Ile; Arg37Lys; Arg37Leu; Arg37Met; Arg37Asn;Arg37Pro; Arg37Gln; Arg37Ser; Arg37Thr; Arg37Val; Arg37Trp; Arg37Tyr;Phe38Ala; Phe38Cys; Phe38Asp; Phe38Glu; Phe38Gly; Phe38His; Phe38Ile;Phe38Lys; Phe38Leu; Phe38Met; Phe38Asn; Phe38Pro; Phe38Gln; Phe38Arg;Phe38Ser; Phe38Thr; Phe38Val; Phe38Trp; Phe38Tyr; Ala39Cys; Ala39Asp;Ala39Glu; Ala39Phe; Ala39Gly; Ala39His; Ala39Ile; Ala39Lys; Ala39Leu;Ala39Met; Ala39Asn; Ala39Pro; Ala39Gln; Ala39Arg; Ala39Ser; Ala39Thr;Ala39Val; Ala39Trp; Ala39Tyr; Phe40Ala; Phe40Cys; Phe40Asp; Phe40Glu;Phe40Gly; Phe40His; Phe40Ile; Phe40Lys; Phe40Leu; Phe40Met; Phe40Asn;Phe40Pro; Phe40Gln; Phe40Arg; Phe40Ser; Phe40Thr; Phe40Val; Phe40Trp;Phe40Tyr; Ala41Cys; Ala41Asp; Ala41Glu; Ala41Phe; Ala41Gly; Ala41His;Ala41Ile; Ala41Lys; Ala41Leu; Ala41Met; Ala41Asn; Ala41Pro; Ala41Gln;Ala41Arg; Ala41Ser; Ala41Thr; Ala41VaI; Ala41Trp; Ala41Tyr; Ser42Ala;Ser42Cys; Ser42Asp; Ser42Glu; Ser42Phe; Ser42Gly; Ser42His; Ser42Ile;Ser42Lys; Ser42Leu; Ser42Met; Ser42Asn; Ser42Pro; Ser42Gln; Ser42Arg;Ser42Thr; Ser42Val; Ser42Trp; Ser42Tyr; Thr43Ala; Thr43Cys; Thr43Asp;Thr43Glu; Thr43Phe; Thr43Gly; Thr43His; Thr43Ile; Thr43Lys; Thr43Leu;Thr43Met; Thr43Asn; Thr43Pro; Thr43Gln; Thr43Arg; Thr43Ser; Thr43Val;Thr43Trp; Thr43Tyr; Ile44Ala; Ile44Cys; Ile44Asp; Ile44Glu; Ile44Phe;Ile44Gly; Ile44His; Ile44Lys; Ile44Leu; Ile44Met; Ile44Asn; Ile44Pro;Ile44Gln; Ile44Arg; Ile44Ser; Ile44Thr; Ile44Val; Ile44Trp; Ile44Tyr;Lys45Ala; Lys45Cys; Lys45Asp; Lys45Glu; Lys45Phe; Lys45Gly; Lys45His;Lys45Ile; Lys45Leu; Lys45Met; Lys45Asn; Lys45Pro; Lys45Gln; Lys45Arg;Lys45Ser; Lys45Thr; Lys45Val; Lys45Trp; Lys45Tyr; Ala46Cys; Ala46Asp;Ala46Glu; Ala46Phe; Ala46Gly; Ala46His; Ala46Ile; Ala46Lys; Ala46Leu;Ala46Met; Ala46Asn; Ala46Pro; Ala46Gln; Ala46Arg; Ala46Ser; Ala46Thr;Ala46Val; Ala46Trp; Ala46Tyr; Leu47Ala; Leu47Cys; Leu47Asp; Leu47Glu;Leu47Phe; Leu47Gly; Leu47His; Leu47Ile; Leu47Lys; Leu47Met; Leu47Asn;Leu47Pro; Leu47Gln; Leu47Arg; Leu47Ser; Leu47Thr; Leu47Val; Leu47Trp;Leu47Tyr; Thr48Ala; Thr48Cys; Thr48Asp; Thr48Glu; Thr48Phe; Thr48Gly;Thr48His; Thr48Ile; Thr48Lys; Thr48Leu; Thr48Met; Thr48Asn; Thr48Pro;Thr48Gln; Thr48Arg; Thr48Ser; Thr48Val; Thr48Trp; Thr48Tyr; Val49Ala;Val49Cys; Val49Asp; Val49Glu; Val49Phe; Val49Gly; Val49His; Val49Ile;Val49Lys; Val49Leu; Val49Met; Val49Asn; Val49Pro; Val49Gln; Val49Arg;Val49Ser; Val49Thr; Val49Trp; Val49Tyr; Gly50Ala; Gly50Cys; Gly50Asp;Gly50Glu; Gly50Phe; Gly50His; Gly50Ile; Gly50Lys; Gly50Leu; Gly50Met;Gly50Asn; Gly50Pro; Gly50Gln; Gly50Arg; Gly50Ser; Gly50Thr; Gly50Val;Gly50Trp; Gly50Tyr; Val51Ala; Val51Cys; Val51Asp; Val51Glu; Val51Phe;Val51Gly; Val51His; Val51Ile; Val51Lys; Val51Leu; Val51Met; Val51Asn;Val51Pro; Val51Gln; Val51Arg; Val51Ser; Val51Thr; Val51Trp; Val51Tyr;Leu52Ala; Leu52Cys; Leu52Asp; Leu52Glu; Leu52Phe; Leu52Gly; Leu52His;Leu52Ile; Leu52Lys; Leu52Met; Leu52Asn; Leu52Pro; Leu52Gln; Leu52Arg;Leu52Ser; Leu52Thr; Leu52Val; Leu52Trp; Leu52Tyr; Leu53Ala; Leu53Cys;Leu53Asp; Leu53Glu; Leu53Phe; Leu53Gly; Leu53His; Leu53Ile; Leu53Lys;Leu53Met; Leu53Asn; Leu53Pro; Leu53Gln; Leu53Arg; Leu53Ser; Leu53Thr;Leu53Val; Leu53Trp; Leu53Tyr; Gln54Ala; Gln54Cys; Gln54Asp; Gln54Glu;Gln54Phe; Gln54Gly; Gln54His; Gln54Ile; Gln54Lys; Gln54Leu; Gln54Met;Gln54Asn; Gln54Pro; Gln54Arg; Gln54Ser; Gln54Thr; Gln54Val; Gln54Trp;Gln54Tyr; Gln55Ala; Gln55Cys; Gln55Asp; Gln55Glu; Gln55Phe; Gln55Gly;Gln55His; Gln55Ile; Gln55Lys; Gln55Leu; Gln55Met; Gln55Asn; Gln55Pro;Gln55Arg; Gln55Ser; Gln55Thr; Gln55Val; Gln55Trp; Gln55Tyr; Lys56Ala;Lys56Cys; Lys56Asp; Lys56Glu; Lys56Phe; Lys56Gly; Lys56His; Lys56Ile;Lys56Leu; Lys56Met; Lys56Asn; Lys56Pro; Lys56Gln; Lys56Arg; Lys56Ser;Lys56Thr; Lys56Val; Lys56Trp; Lys56Tyr; Ser57Ala; Ser57Cys; Ser57Asp;Ser57Glu; Ser57Phe; Ser57Gly; Ser57His; Ser57Ile; Ser57Lys; Ser57Leu;Ser57Met; Ser57Asn; Ser57Pro; Ser57Gln; Ser57Arg; Ser57Thr; Ser57Val;Ser57Trp; Ser57Tyr; Ile58Ala; Ile58Cys; Ile58Asp; Ile58Glu; Ile58Phe;Ile58Gly; Ile58His; Ile58Lys; Ile58Leu; Ile58Met; Ile58Asn; Ile58Pro;Ile58Gln; Ile58Arg; Ile58Ser; Ile58Thr; Ile58Val; Ile58Trp; Ile58Tyr;Glu59Ala; Glu59Cys; Glu59Asp; Glu59Phe; Glu59Gly; Glu59His; Glu59Ile;Glu59Lys; Glu59Leu; Glu59Met; Glu59Asn; Glu59Pro; Glu59Gln; Glu59Arg;Glu59Ser; Glu59Thr; Glu59Val; Glu59Trp; Glu59Tyr; Asp60Ala; Asp60Cys;Asp60Glu; Asp60Phe; Asp60Gly; Asp60His; Asp60Ile; Asp60Lys; Asp60Leu;Asp60Met; Asp60Asn; Asp60Pro; Asp60Gln; Asp60Arg; Asp60Ser; Asp60Thr;Asp60Val; Asp60Trp; Asp60Tyr; Leu61Ala; Leu61Cys; Leu61Asp; Leu61Glu;Leu61Phe; Leu61Gly; Leu61His; Leu61Ile; Leu61Lys; Leu61Met; Leu61Asn;Leu61Pro; Leu61Gln; Leu61Arg; Leu61Ser; Leu61Thr; Leu61Val; Leu61Trp;Leu61Tyr; Asn62Ala; Asn62Cys; Asn62Asp; Asn62Glu; Asn62Phe; Asn62Gly;Asn62His; Asn62Ile; Asn62Lys; Asn62Leu; Asn62Met; Asn62Pro; Asn62Gln;Asn62Arg; Asn62Ser; Asn62Thr; Asn62Val; Asn62Trp; Asn62Tyr; Gln63Ala;Gln63Cys; Gln63Asp; Gln63Glu; Gln63Phe; Gln63Gly; Gln63His; Gln63Ile;Gln63Lys; Gln63Leu; Gln63Met; Gln63Asn; Gln63Pro; Gln63Arg; Gln63Ser;Gln63Thr; Gln63Val; Gln63Trp; Gln63Tyr; Arg64Ala; Arg64Cys; Arg64Asp;Arg64Glu; Arg64Phe; Arg64Gly; Arg64His; Arg64Ile; Arg64Lys; Arg64Leu;Arg64Met; Arg64Asn; Arg64Pro; Arg64Gln; Arg64Ser; Arg64Thr; Arg64Val;Arg64Trp; Arg64Tyr; Ile65Ala; IIe65Cys; IIe65Asp; IIe65Glu; IIe65Phe;IIe65Gly; IIe65His; IIe65Lys; IIe65Leu; IIe65Met; IIe65Asn; IIe65Pro;IIe65Gln; IIe65Arg; IIe65Ser; IIe65Thr; Ile65Val; IIe65Trp; IIe65Tyr;Thr66Ala; Thr66Cys; Thr66Asp; Thr66Glu; Thr66Phe; Thr66Gly; Thr66His;Thr66Ile; Thr66Lys; Thr66Leu; Thr66Met; Thr66Asn; Thr66Pro; Thr66Gln;Thr66Arg; Thr66Ser; Thr66Val; Thr66Trp; Thr66Tyr; Tyr67Ala; Tyr67Cys;Tyr67Asp; Tyr67Glu; Tyr67Phe; Tyr67Gly; Tyr67His; Tyr67Ile; Tyr67Lys;Tyr67Leu; Tyr67Met; Tyr67Asn; Tyr67Pro; Tyr67Gln; Tyr67Arg; Tyr67Ser;Tyr67Thr; Tyr67Val; Tyr67Trp; Thr68Ala; Thr68Cys; Thr68Asp; Thr68Glu;Thr68Phe; Thr68Gly; Thr68His; Thr68Ile; Thr68Lys; Thr68Leu; Thr68Met;Thr68Asn; Thr68Pro; Thr68Gln; Thr68Arg; Thr68Ser; Thr68Val; Thr68Trp;Thr68Tyr; Arg69Ala; Arg69Cys; Arg69Asp; Arg69Glu; Arg69Phe; Arg69Gly;Arg69His; Arg69Ile; Arg69Lys; Arg69Leu; Arg69Met; Arg69Asn; Arg69Pro;Arg69Gln; Arg69Ser; Arg69Thr; Arg69Val; Arg69Trp; Arg69Tyr; Asp70Ala;Asp70Cys; Asp70Glu; Asp70Phe; Asp70Gly; Asp70His; Asp70Ile; Asp70Lys;Asp70Leu; Asp70Met; Asp70Asn; Asp70Pro; Asp70Gln; Asp70Arg; Asp70Ser;Asp70Thr; Asp70Val; Asp70Trp; Asp70Tyr; Asp71Ala; Asp71Cys; Asp71Glu;Asp71Phe; Asp71Gly; Asp71His; Asp71Ile; Asp71Lys; Asp71Leu; Asp71Met;Asp71Asn; Asp71Pro; Asp71Gln; Asp71Arg; Asp71Ser; Asp71Thr; Asp71Val;Asp71Trp; Asp71Tyr; Leu72Ala; Leu72Cys; Leu72Asp; Leu72Glu; Leu72Phe;Leu72Gly; Leu72His; Leu72Ile; Leu72Lys; Leu72Met; Leu72Asn; Leu72Pro;Leu72Gln; Leu72Arg; Leu72Ser; Leu72Thr; Leu72Val; Leu72Trp; Leu72Tyr;Val73Ala; Val73Cys; Val73Asp; Val73Glu; Val73Phe; Val73Gly; Val73His;Val73Ile; Val73Lys; Val73Leu; Val73Met; Val73Asn; Val73Pro; Val73Gln;Val73Arg; Val73Ser; Val73Thr; Val73Trp; Val73Tyr; Asn74Ala; Asn74Cys;Asn74Asp; Asn74Glu; Asn74Phe; Asn74Gly; Asn74His; Asn74Ile; Asn74Lys;Asn74Leu; Asn74Met; Asn74Pro; Asn74Gln; Asn74Arg; Asn74Ser; Asn74Thr;Asn74Val; Asn74Trp; Asn74Tyr; Tyr75Ala; Tyr75Cys; Tyr75Asp; Tyr75Glu;Tyr75Phe; Tyr75Gly; Tyr75His; Tyr75Ile; Tyr75Lys; Tyr75Leu; Tyr75Met;Tyr75Asn; Tyr75Pro; Tyr75Gln; Tyr75Arg; Tyr75Ser; Tyr75Thr; Tyr75Val;Tyr75Trp; Asn76Ala; Asn76Cys; Asn76Asp; Asn76Glu; Asn76Phe; Asn76Gly;Asn76His; Asn76Ile; Asn76Lys; Asn76Leu; Asn76Met; Asn76Pro; Asn76Gln;Asn76Arg; Asn76Ser; Asn76Thr; Asn76Val; Asn76Trp; Asn76Tyr; Pro77Ala;Pro77Cys; Pro77Asp; Pro77Glu; Pro77Phe; Pro77Gly; Pro77His; Pro77Ile;Pro77Lys; Pro77Leu; Pro77Met; Pro77Asn; Pro77Gln; Pro77Arg; Pro77Ser;Pro77Thr; Pro77Val; Pro77Trp; Pro77Tyr; IIe78Ala; IIe78Cys; IIe78Asp;Ile78Glu; IIe78Phe; Ile78Gly; IIe78His; IIe78Lys; IIe78Leu; IIe78Met;IIe78Asn; IIe78Pro; IIe78Gln; IIe78Arg; IIe78Ser; IIe78Thr; IIe78VaI;IIe78Trp; IIe78Tyr; Thr79Ala; Thr79Cys; Thr79Asp; Thr79Glu; Thr79Phe;Thr79Gly; Thr79His; Thr79Ile; Thr79Lys; Thr79Leu; Thr79Met; Thr79Asn;Thr79Pro; Thr79Gln; Thr79Arg; Thr79Ser; Thr79VaI; Thr79Trp; Thr79Tyr;Glu80Ala; Glu80Cys; Glu80Asp; Glu80Phe; Glu80Gly; Glu80His; Glu80Ile;Glu80Lys; Glu80Leu; Glu80Met; Glu80Asn; Glu80Pro; Glu80Gln; Glu80Arg;Glu80Ser; Glu80Thr; Glu80Val; Glu80Trp; Glu80Tyr; Lys81Ala; Lys81Cys;Lys81Asp; Lys81Glu; Lys81Phe; Lys81Gly; Lys81His; Lys81Ile; Lys81Leu;Lys81Met; Lys81Asn; Lys81Pro; Lys81Gln; Lys81Arg; Lys81Ser; Lys81Thr;Lys81Val; Lys81Trp; Lys81Tyr; His82Ala; His82Cys; His82Asp; His82Glu;His82Phe; His82Gly; His82Ile; His82Lys; His82Leu; His82Met; His82Asn;His82Pro; His82Gln; His82Arg; His82Ser; His82Thr; His82Val; His82Trp;His82Tyr; Val83Ala; Val83Cys; Val83Asp; Val83Glu; Val83Phe; Val83Gly;Val83His; Val83Ile; Val83Lys; Val83Leu; Val83Met; Val83Asn; Val83Pro;Val83Gln; Val83Arg; Val83Ser; Val83Thr; Val83Trp; Val83Tyr; Asp84Ala;Asp84Cys; Asp84Glu; Asp84Phe; Asp84Gly; Asp84His; Asp84Ile; Asp84Lys;Asp84Leu; Asp84Met; Asp84Asn; Asp84Pro; Asp84Gln; Asp84Arg; Asp84Ser;Asp84Thr; Asp84Val; Asp84Trp; Asp84Tyr; Thr85Ala; Thr85Cys; Thr85Asp;Thr85Glu; Thr85Phe; Thr85Gly; Thr85His; Thr85Ile; Thr85Lys; Thr85Leu;Thr85Met; Thr85Asn; Thr85Pro; Thr85Gln; Thr85Arg; Thr85Ser; Thr85Val;Thr85Trp; Thr85Tyr; Gly86Ala; Gly86Cys; Gly86Asp; Gly86Glu; Gly86Phe;Gly86His; Gly86Ile; Gly86Lys; Gly86Leu; Gly86Met; Gly86Asn; Gly86Pro;Gly86Gln; Gly86Arg; Gly86Ser; Gly86Thr; Gly86Val; Gly86Trp; Gly86Tyr;Met87Ala; Met87Cys; Met87Asp; Met87Glu; Met87Phe; Met87Gly; Met87His;Met87Ile; Met87Lys; Met87Leu; Met87Asn; Met87Pro; Met87Gln; Met87Arg;Met87Ser; Met87Thr; Met87Val; Met87Trp; Met87Tyr; Thr88Ala; Thr88Cys;Thr88Asp; Thr88Glu; Thr88Phe; Thr88Gly; Thr88His; Thr88Ile; Thr88Lys;Thr88Leu; Thr88Met; Thr88Asn; Thr88Pro; Thr88Gln; Thr88Arg; Thr88Ser;Thr88Val; Thr88Trp; Thr88Tyr; Leu89Ala; Leu89Cys; Leu89Asp; Leu89Glu;Leu89Phe; Leu89Gly; Leu89His; Leu89Ile; Leu89Lys; Leu89Met; Leu89Asn;Leu89Pro; Leu89Gln; Leu89Arg; Leu89Ser; Leu89Thr; Leu89Val; Leu89Trp;Leu89Tyr; Lys90Ala; Lys90Cys; Lys90Asp; Lys90Glu; Lys90Phe; Lys90Gly;Lys90His; Lys90Ile; Lys90Leu; Lys90Met; Lys90Asn; Lys90Pro; Lys90Gln;Lys90Arg; Lys90Ser; Lys90Thr; Lys90Val; Lys90Trp; Lys90Tyr; Glu91Ala;Glu91Cys; Glu91Asp; Glu91Phe; Glu91Gly; Glu91His; Glu91Ile; Glu91Lys;Glu91Leu; Glu91Met; Glu91Asn; Glu91Pro; Glu91Gln; Glu91Arg; Glu91Ser;Glu91Thr; Glu91Val; Glu91Trp; Glu91Tyr; Leu92Ala; Leu92Cys; Leu92Asp;Leu92Glu; Leu92Phe; Leu92Gly; Leu92His; Leu92Ile; Leu92Lys; Leu92Met;Leu92Asn; Leu92Pro; Leu92Gln; Leu92Arg; Leu92Ser; Leu92Thr; Leu92Val;Leu92Trp; Leu92Tyr; Ala93Cys; Ala93Asp; Ala93Glu; Ala93Phe; Ala93Gly;Ala93His; Ala93Ile; Ala93Lys; Ala93Leu; Ala93Met; Ala93Asn; Ala93Pro;Ala93Gln; Ala93Arg; Ala93Ser; Ala93Thr; Ala93Val; Ala93Trp; Ala93Tyr;Asp94Ala; Asp94Cys; Asp94Glu; Asp94Phe; Asp94Gly; Asp94His; Asp94Ile;Asp94Lys; Asp94Leu; Asp94Met; Asp94Asn; Asp94Pro; Asp94Gln; Asp94Arg;Asp94Ser; Asp94Thr; Asp94Val; Asp94Trp; Asp94Tyr; Ala95Cys; Ala95Asp;Ala95Glu; Ala95Phe; Ala95Gly; Ala95His; Ala95Ile; Ala95Lys; Ala95Leu;Ala95Met; Ala95Asn; Ala95Pro; Ala95Gln; Ala95Arg; Ala95Ser; Ala95Thr;Ala95Val; Ala95Trp; Ala95Tyr; Ser96Ala; Ser96Cys; Ser96Asp; Ser96Glu;Ser96Phe; Ser96Gly; Ser96His; Ser96Ile; Ser96Lys; Ser96Leu; Ser96Met;Ser96Asn; Ser96Pro; Ser96Gln; Ser96Arg; Ser96Thr; Ser96Val; Ser96Trp;Ser96Tyr; Leu97Ala; Leu97Cys; Leu97Asp; Leu97Glu; Leu97Phe; Leu97Gly;Leu97His; Leu97Ile; Leu97Lys; Leu97Met; Leu97Asn; Leu97Pro; Leu97Gln;Leu97Arg; Leu97Ser; Leu97Thr; Leu97Val; Leu97Trp; Leu97Tyr; Arg98Ala;Arg98Cys; Arg98Asp; Arg98Glu; Arg98Phe; Arg98Gly; Arg98His; Arg98Ile;Arg98Lys; Arg98Leu; Arg98Met; Arg98Asn; Arg98Pro; Arg98Gln; Arg98Ser;Arg98Thr; Arg98Val; Arg98Trp; Arg98Tyr; Tyr99Ala; Tyr99Cys; Tyr99Asp;Tyr99Glu; Tyr99Phe; Tyr99Gly; Tyr99His; Tyr99Ile; Tyr99Lys; Tyr99Leu;Tyr99Met; Tyr99Asn; Tyr99Pro; Tyr99Gln; Tyr99Arg; Tyr99Ser; Tyr99Thr;Tyr99Val; Tyr99Trp; Ser100Ala; Ser100Cys; Ser100Asp; Ser100Glu;Ser100Phe; Ser100Gly; Ser100His; Ser100Ile; Ser100Lys; Ser100Leu;Ser100Met; Ser100Asn; Ser100Pro; Ser100Gln; Ser100Arg; Ser100Thr;Ser100Val; Ser100Trp; Ser100Tyr; Asp101Ala; Asp101Cys; Asp101Glu;Asp101Phe; Asp101Gly; Asp101His; Asp101Ile; Asp101Lys; Asp101Leu;Asp101Met; Asp101Asn; Asp101Pro; Asp101Gln; Asp101Arg; Asp101Ser;Asp101Thr; Asp101Val; Asp101Trp; Asp101Tyr; Asn102Ala; Asn102Cys;Asn102Asp; Asn102Glu; Asn102Phe; Asn102Gly; Asn102His; Asn102Ile;Asn102Lys; Asn102Leu; Asn102Met; Asn102Pro; Asn102Gln; Asn102Arg;Asn102Ser; Asn102Thr; Asn102Val; Asn102Trp; Asn102Tyr; Ala103Cys;Ala103Asp; Ala103Glu; Ala103Phe; Ala103Gly; Ala103His; Ala103Ile;Ala103Lys; Ala103Leu; Ala103Met; Ala103Asn; Ala103Pro; Ala103Gln;Ala103Arg; Ala103Ser; Ala103Thr; Ala103Val; Ala103Trp; Ala103Tyr;Ala104Cys; Ala104Asp; Ala104Glu; Ala104Phe; Ala104Gly; Ala104His;Ala104Ile; Ala104Lys; Ala104Leu; Ala104Met; Ala104Asn; Ala104Pro;Ala104Gln; Ala104Arg; Ala104Ser; Ala104Thr; Ala104Val; Ala104Trp;Ala104Tyr; Gln105Ala; Gln105Cys; Gln105Asp; Gln105Glu; Gln105Phe;Gln105Gly; Gln105His; Gln105Ile; Gln105Lys; Gln105Leu; Gln105Met;Gln105Asn; Gln105Pro; Gln105Arg; Gln105Ser; Gln105Thr; Gln105Val;Gln105Trp; Gln105Tyr; Asn106Ala; Asn106Cys; Asn106Asp; Asn106Glu;Asn106Phe; Asn106Gly; Asn106His; Asn106Ile; Asn106Lys; Asn106Leu;Asn106Met; Asn106Pro; Asn106Gln; Asn106Arg; Asn106Ser; Asn106Thr;Asn106Val; Asn106Trp; Asn106Tyr; Leu107Ala; Leu107Cys; Leu107Asp;Leu107Glu; Leu107Phe; Leu107Gly; Leu107His; Leu107Ile; Leu107Lys;Leu107Met; Leu107Asn; Leu107Pro; Leu107Gln; Leu107Arg; Leu107Ser;Leu107Thr; Leu107Val; Leu107Trp; Leu107Tyr; Ile108Ala; Ile108Cys;Ile108Asp; Ile108Glu; Ile108Phe; Ile108Gly; Ile108His; Ile108Lys;Ile108Leu; Ile108Met; Ile108Asn; Ile108Pro; Ile108Gln; Ile108Arg;Ile108Ser; Ile108Thr; Ile108Val; Ile108Trp; Ile108Tyr; Leu109Ala;Leu109Cys; Leu109Asp; Leu109Glu; Leu109Phe; Leu109Gly; Leu109His;Leu109Ile; Leu109Lys; Leu109Met; Leu109Asn; Leu109Pro; Leu109Gln;Leu109Arg; Leu109Ser; Leu109Thr; Leu109Val; Leu109Trp; Leu109Tyr;Lys110Ala; Lys110Cys; Lys110Asp; Lys110Glu; Lys110Phe; Lys110Gly;Lys110His; Lys110Ile; Lys110Leu; Lys110Met; Lys110Asn; Lys110Pro;Lys110Gln; Lys110Arg; Lys110Ser; Lys110Thr; Lys110Val; Lys110Trp;Lys110Tyr; Gln111Ala; Gln111Cys; Gln111Asp; Gln111Glu; Gln111Phe;Gln111Gly; Gln111His; Gln111Ile; Gln111Lys; Gln111Leu; Gln111Met;Gln111Asn; Gln111Pro; Gln111Arg; Gln111Ser; Gln111Thr; Gln111Val;Gln111Trp; Gln111Tyr; Ile112Ala; Ile112Cys; Ile112Asp; Ile112Glu;Ile112Phe; Ile112Gly; Ile112His; Ile112Lys; Ile112Leu; Ile112Met;Ile112Asn; Ile112Pro; Ile112Gln; Ile112Arg; Ile112Ser; Ile112Thr;Ile112Val; Ile112Trp; Ile112Tyr; Gly113Ala; Gly113Cys; Gly113Asp;Gly113Glu; Gly113Phe; Gly113His; Gly113Ile; Gly113Lys; Gly113Leu;Gly113Met; Gly113Asn; Gly113Pro; Gly113Gln; Gly113Arg; Gly113Ser;Gly113Thr; Gly113Val; Gly113Trp; Gly113Tyr; Gly114Ala; Gly114Cys;Gly114Asp; Gly114Glu; Gly114Phe; Gly114His; Gly114Ile; Gly114Lys;Gly114Leu; Gly114Met; Gly114Asn; Gly114Pro; Gly114Gln; Gly114Arg;Gly114Ser; Gly114Thr; Gly114Val; Gly114Trp; Gly114Tyr; Pro115Ala;Pro115Cys; Pro115Asp; Pro115Glu; Pro115Phe; Pro115Gly; Pro115His;Pro115Ile; Pro115Lys; Pro115Leu; Pro115Met; Pro115Asn; Pro115Gln;Pro115Arg; Pro115Ser; Pro115Thr; Pro115Val; Pro115Trp; Pro115Tyr;Glu116Ala; Glu116Cys; Glu116Asp; Glu116Phe; Glu116Gly; Glu116His;Glu116Ile; Glu116Lys; Glu116Leu; Glu116Met; Glu116Asn; Glu116Pro;Glu116Gln; Glu116Arg; Glu116Ser; Glu116Thr; Glu116Val; Glu116Trp;Glu116Tyr; Ser117Ala; Ser117Cys; Ser117Asp; Ser117Glu; Ser117Phe;Ser117Gly; Ser117His; Ser117Ile; Ser117Lys; Ser117Leu; Ser117Met;Ser117Asn; Ser117Pro; Ser117Gln; Ser117Arg; Ser117Thr; Ser117Val;Ser117Trp; Ser117Tyr; Leu118Ala; Leu118Cys; Leu118Asp; Leu118Glu;Leu118Phe; Leu118Gly; Leu118His; Leu118Ile; Leu118Lys; Leu118Met;Leu118Asn; Leu118Pro; Leu118Gln; Leu118Arg; Leu118Ser; Leu118Thr;Leu118Val; Leu118Trp; Leu118Tyr; Lys119Ala; Lys119Cys; Lys119Asp;Lys119Glu; Lys119Phe; Lys119Gly; Lys119His; Lys119Ile; Lys119Leu;Lys119Met; Lys119Asn; Lys119Pro; Lys119Gln; Lys119Arg; Lys119Ser;Lys119Thr; Lys119Val; Lys119Trp; Lys119Tyr; Lys120Ala; Lys120Cys;Lys120Asp; Lys120Glu; Lys120Phe; Lys120Gly; Lys120His; Lys120Ile;Lys120Leu; Lys120Met; Lys120Asn; Lys120Pro; Lys120Gln; Lys120Arg;Lys120Ser; Lys120Thr; Lys120Val; Lys120Trp; Lys120Tyr; Glu121Ala;Glu121Cys; Glu121Asp; Glu121Phe; Glu121Gly; Glu121His; Glu121Ile;Glu121Lys; Glu121Leu; Glu121Met; Glu121Asn; Glu121Pro; Glu121Gln;Glu121Arg; Glu121Ser; Glu121Thr; Glu121Val; Glu121Trp; Glu121Tyr;Leu122Ala; Leu122Cys; Leu122Asp; Leu122Glu; Leu122Phe; Leu122Gly;Leu122His; Leu122Ile; Leu122Lys; Leu122Met; Leu122Asn; Leu122Pro;Leu122Gln; Leu122Arg; Leu122Ser; Leu122Thr; Leu122Val; Leu122Trp;Leu122Tyr; Arg123Ala; Arg123Cys; Arg123Asp; Arg123Glu; Arg123Phe;Arg123Gly; Arg123His; Arg123Ile; Arg123Lys; Arg123Leu; Arg123Met;Arg123Asn; Arg123Pro; Arg123Gln; Arg123Ser; Arg123Thr; Arg123Val;Arg123Trp; Arg123Tyr; Lys124Ala; Lys124Cys; Lys124Asp; Lys124Glu;Lys124Phe; Lys124Gly; Lys124His; Lys124Ile; Lys124Leu; Lys124Met;Lys124Asn; Lys124Pro; Lys124Gln; Lys124Arg; Lys124Ser; Lys124Thr;Lys124Val; Lys124Trp; Lys124Tyr; Ile125Ala; Ile125Cys; Ile125Asp;Ile125Glu; Ile125Phe; Ile125Gly; Ile125His; Ile125Lys; Ile125Leu;Ile125Met; Ile125Asn; Ile125Pro; Ile125Gln; Ile125Arg; Ile125Ser;Ile125Thr; Ile125Val; Ile125Trp; Ile125Tyr; Gly126Ala; Gly126Cys;Gly126Asp; Gly126Glu; Gly126Phe; Gly126His; Gly126Ile; Gly126Lys;Gly126Leu; Gly126Met; Gly126Asn; Gly126Pro; Gly126Gln; Gly126Arg;Gly126Ser; Gly126Thr; Gly126Val; Gly126Trp; Gly126Tyr; Asp127Ala;Asp127Cys; Asp127Glu; Asp127Phe; Asp127Gly; Asp127His; Asp127Ile;Asp127Lys; Asp127Leu; Asp127Met; Asp127Asn; Asp127Pro; Asp127Gln;Asp127Arg; Asp127Ser; Asp127Thr; Asp127Val; Asp127Trp; Asp127Tyr;Glu128Ala; Glu128Cys; Glu128Asp; Glu128Phe; Glu128Gly; Glu128His;Glu128Ile; Glu128Lys; Glu128Leu; Glu128Met; Glu128Asn; Glu128Pro;Glu128Gln; Glu128Arg; Glu128Ser; Glu128Thr; Glu128Val; Glu128Trp;Glu128Tyr; Val129Ala; Val129Cys; Val129Asp; Val129Glu; Val129Phe;Val129Gly; Val129His; Val129Ile; Val129Lys; Val129Leu; Val129Met;Val129Asn; Val129Pro; Val129Gln; Val129Arg; Val129Ser; Val129Thr;Val129Trp; Val129Tyr; Thr130Ala; Thr130Cys; Thr130Asp; Thr130Glu;Thr130Phe; Thr130Gly; Thr130His; Thr130Ile; Thr130Lys; Thr130Leu;Thr130Met; Thr130Asn; Thr130Pro; Thr130Gln; Thr130Arg; Thr130Ser;Thr130Val; Thr130Trp; Thr130Tyr; Asn131Ala; Asn131Cys; Asn131Asp;Asn131Glu; Asn131Phe; Asn131Gly; Asn131His; Asn131Ile; Asn131Lys;Asn131Leu; Asn131Met; Asn131Pro; Asn131Gln; Asn131Arg; Asn131Ser;Asn131Thr; Asn131Val; Asn131Trp; Asn131Tyr; Pro132Ala; Pro132Cys;Pro132Asp; Pro132Glu; Pro132Phe; Pro132Gly; Pro132His; Pro132Ile;Pro132Lys; Pro132Leu; Pro132Met; Pro132Asn; Pro132Gln; Pro132Arg;Pro132Ser; Pro132Thr; Pro132Val; Pro132Trp; Pro132Tyr; Glu133Ala;Glu133Cys; Glu133Asp; Glu133Phe; Glu133Gly; Glu133His; Glu133Ile;Glu133Lys; Glu133Leu; Glu133Met; Glu133Asn; Glu133Pro; Glu133Gln;Glu133Arg; Glu133Ser; Glu133Thr; Glu133Val; Glu133Trp; Glu133Tyr;Arg134Ala; Arg134Cys; Arg134Asp; Arg134Glu; Arg134Phe; Arg134Gly;Arg134His; Arg134Ile; Arg134Lys; Arg134Leu; Arg134Met; Arg134Asn;Arg134Pro; Arg134Gln; Arg134Ser; Arg134Thr; Arg134Val; Arg134Trp;Arg134Tyr; Phe135Ala; Phe135Cys; Phe135Asp; Phe135Glu; Phe135Gly;Phe135His; Phe135Ile; Phe135Lys; Phe135Leu; Phe135Met; Phe135Asn;Phe135Pro; Phe135Gln; Phe135Arg; Phe135Ser; Phe135Thr; Phe135Val;Phe135Trp; Phe135Tyr; Glu136Ala; Glu136Cys; Glu136Asp; Glu136Phe;Glu136Gly; Glu136His; Glu136Ile; Glu136Lys; Glu136Leu; Glu136Met;Glu136Asn; Glu136Pro; Glu136Gln; Glu136Arg; Glu136Ser; Glu136Thr;Glu136Val; Glu136Trp; Glu136Tyr; Pro137Ala; Pro137Cys; Pro137Asp;Pro137Glu; Pro137Phe; Pro137Gly; Pro137His; Pro137Ile; Pro137Lys;Pro137Leu; Pro137Met; Pro137Asn; Pro137Gln; Pro137Arg; Pro137Ser;Pro137Thr; Pro137Val; Pro137Trp; Pro137Tyr; Glu138Ala; Glu138Cys;Glu138Asp; Glu138Phe; Glu138Gly; Glu138His; Glu138Ile; Glu138Lys;Glu138Leu; Glu138Met; Glu138Asn; Glu138Pro; Glu138Gln; Glu138Arg;Glu138Ser; Glu138Thr; Glu138Val; Glu138Trp; Glu138Tyr; Leu139Ala;Leu139Cys; Leu139Asp; Leu139Glu; Leu139Phe; Leu139Gly; Leu139His;Leu139Ile; Leu139Lys; Leu139Met; Leu139Asn; Leu139Pro; Leu139Gln;Leu139Arg; Leu139Ser; Leu139Thr; Leu139Val; Leu139Trp; Leu139Tyr;Asn140Ala; Asn140Cys; Asn140Asp; Asn140Glu; Asn140Phe; Asn140Gly;Asn140His; Asn140Ile; Asn140Lys; Asn140Leu; Asn140Met; Asn140Pro;Asn140Gln; Asn140Arg; Asn140Ser; Asn140Thr; Asn140Val; Asn140Trp;Asn140Tyr; Glu141Ala; Glu141Cys; Glu141Asp; Glu141Phe; Glu141Gly;Glu141His; Glu141Ile; Glu141Lys; Glu141Leu; Glu141Met; Glu141Asn;Glu141Pro; Glu141Gln; Glu141Arg; Glu141Ser; Glu141Thr; Glu141Val;Glu141Trp; Glu141Tyr; Val142Ala; Val142Cys; Val142Asp; Val142Glu;Val142Phe; Val142Gly; Val142His; Val142Ile; Val142Lys; Val142Leu;Val142Met; Val142Asn; Val142Pro; Val142Gln; Val142Arg; Val142Ser;Val142Thr; Val142Trp; Val142Tyr; Asn143Ala; Asn143Cys; Asn143Asp;Asn143Glu; Asn143Phe; Asn143Gly; Asn143His; Asn143Ile; Asn143Lys;Asn143Leu; Asn143Met; Asn143Pro; Asn143Gln; Asn143Arg; Asn143Ser;Asn143Thr; Asn143Val; Asn143Trp; Asn143Tyr; Pro144Ala; Pro144Cys;Pro144Asp; Pro144Glu; Pro144Phe; Pro144Gly; Pro144His; Pro144Ile;Pro144Lys; Pro144Leu; Pro144Met; Pro144Asn; Pro144Gln; Pro144Arg;Pro144Ser; Pro144Thr; Pro144Val; Pro144Trp; Pro144Tyr; Gly145Ala;Gly145Cys; Gly145Asp; Gly145Glu; Gly145Phe; Gly145His; Gly145Ile;Gly145Lys; Gly145Leu; Gly145Met; Gly145Asn; Gly145Pro; Gly145Gln;Gly145Arg; Gly145Ser; Gly145Thr; Gly145Val; Gly145Trp; Gly145Tyr;Glu146Ala; Glu146Cys; Glu146Asp; Glu146Phe; Glu146Gly; Glu146His;Glu146Ile; Glu146Lys; Glu146Leu; Glu146Met; Glu146Asn; Glu146Pro;Glu146Gln; Glu146Arg; Glu146Ser; Glu146Thr; Glu146Val; Glu146Trp;Glu146Tyr; Thr147Ala; Thr147Cys; Thr147Asp; Thr147Glu; Thr147Phe;Thr147Gly; Thr147His; Thr147Ile; Thr147Lys; Thr147Leu; Thr147Met;Thr147Asn; Thr147Pro; Thr147Gln; Thr147Arg; Thr147Ser; Thr147Val;Thr147Trp; Thr147Tyr; Gln148Ala; Gln148Cys; Gln148Asp; Gln148Glu;Gln148Phe; Gln148Gly; Gln148His; Gln148Ile; Gln148Lys; Gln148Leu;Gln148Met; Gln148Asn; Gln148Pro; Gln148Arg; Gln148Ser; Gln148Thr;Gln148Val; Gln148Trp; Gln148Tyr; Asp149Ala; Asp149Cys; Asp149Glu;Asp149Phe; Asp149Gly; Asp149His; Asp149Ile; Asp149Lys; Asp149Leu;Asp149Met; Asp149Asn; Asp149Pro; Asp149Gln; Asp149Arg; Asp149Ser;Asp149Thr; Asp149Val; Asp149Trp; Asp149Tyr; Thr150Ala; Thr150Cys;Thr150Asp; Thr150Glu; Thr150Phe; Thr150Gly; Thr150His; Thr150Ile;Thr150Lys; Thr150Leu; Thr150Met; Thr150Asn; Thr150Pro; Thr150Gln;Thr150Arg; Thr150Ser; Thr150Val; Thr150Trp; Thr150Tyr; Ser151Ala;Ser151Cys; Ser151Asp; Ser151Glu; Ser151Phe; Ser151Gly; Ser151His;Ser151Ile; Ser151Lys; Ser151Leu; Ser151Met; Ser151Asn; Ser151Pro;Ser151Gln; Ser151Arg; Ser151Thr; Ser151Val; Ser151Trp; Ser151Tyr;Thr152Ala; Thr152Cys; Thr152Asp; Thr152Glu; Thr152Phe; Thr152Gly;Thr152His; Thr152Ile; Thr152Lys; Thr152Leu; Thr152Met; Thr152Asn;Thr152Pro; Thr152Gln; Thr152Arg; Thr152Ser; Thr152Val; Thr152Trp;Thr152Tyr; Ala153Cys; Ala153Asp; Ala153Glu; Ala153Phe; Ala153Gly;Ala153His; Ala153Ile; Ala153Lys; Ala153Leu; Ala153Met; Ala153Asn;Ala153Pro; Ala153Gln; Ala153Arg; Ala153Ser; Ala153Thr; Ala153Val;Ala153Trp; Ala153Tyr; Arg154Ala; Arg154Cys; Arg154Asp; Arg154Glu;Arg154Phe; Arg154Gly; Arg154His; Arg154Ile; Arg154Lys; Arg154Leu;Arg154Met; Arg154Asn; Arg154Pro; Arg154Gln; Arg154Ser; Arg154Thr;Arg154Val; Arg154Trp; Arg154Tyr; Ala155Cys; Ala155Asp; Ala155Glu;Ala155Phe; Ala155Gly; Ala155His; Ala155Ile; Ala155Lys; Ala155Leu;Ala155Met; Ala155Asn; Ala155Pro; Ala155Gln; Ala155Arg; Ala155Ser;Ala155Thr; Ala155Val; Ala155Trp; Ala155Tyr; Leu156Ala; Leu156Cys;Leu156Asp; Leu156Glu; Leu156Phe; Leu156Gly; Leu156His; Leu156Ile;Leu156Lys; Leu156Met; Leu156Asn; Leu156Pro; Leu156Gln; Leu156Arg;Leu156Ser; Leu156Thr; Leu156Val; Leu156Trp; Leu156Tyr; Val157Ala;Val157Cys; Val157Asp; Val157Glu; Val157Phe; Val157Gly; Val157His;Val157Ile; Val157Lys; Val157Leu; Val157Met; Val157Asn; Val157Pro;Val157Gln; Val157Arg; Val157Ser; Val157Thr; Val157Trp; Val157Tyr;Thr158Ala; Thr158Cys; Thr158Asp; Thr158Glu; Thr158Phe; Thr158Gly;Thr158His; Thr158Ile; Thr158Lys; Thr158Leu; Thr158Met; Thr158Asn;Thr158Pro; Thr158Gln; Thr158Arg; Thr158Ser; Thr158Val; Thr158Trp;Thr158Tyr; Seri 59Ala; Ser159Cys; Ser159Asp; Ser159Glu; Ser159Phe;Ser159Gly; Ser159His; Ser159Ile; Ser159Lys; Ser159Leu; Ser159Met;Ser159Asn; Ser159Pro; Ser159Gln; Ser159Arg; Ser159Thr; Ser159Val;Ser159Trp; Ser159Tyr; Leu160Ala; Leu160Cys; Leu160Asp; Leu160Glu;Leu160Phe; Leu160Gly; Leu160His; Leu 160Ile; Leu160Lys; Leu160Met;Leu160Asn; Leu160Pro; Leu160Gln; Leu160Arg; Leu160Ser; Leu160Thr;Leu160Val; Leu160Trp; Leu160Tyr; Arg161Ala; Arg161Cys; Arg161Asp;Arg161Glu; Arg161Phe; Arg161Gly; Arg161His; Arg161Ile; Arg161Lys;Arg161Leu; Arg161Met; Arg161Asn; Arg161Pro; Arg161Gln; Arg161Ser;Arg161Thr; Arg161Val; Arg161Trp; Arg161Tyr; Ala162Cys; Ala162Asp;Ala162Glu; Ala162Phe; Ala162Gly; Ala162His; Ala162Ile; Ala162Lys;Ala162Leu; Ala162Met; Ala162Asn; Ala162Pro; Ala162Gln; Ala162Arg;Ala162Ser; Ala162Thr; Ala162Val; Ala162Trp; Ala162Tyr; Phe163Ala;Phe163Cys; Phe163Asp; Phe163Glu; Phe163Gly; Phe163His; Phe163Ile;Phe163Lys; Phe163Leu; Phe163Met; Phe163Asn; Phe163Pro; Phe163Gln;Phe163Arg; Phe163Ser; Phe163Thr; Phe163Val; Phe163Trp; Phe163Tyr;Ala164Cys; Ala164Asp; Ala164Glu; Ala164Phe; Ala164Gly; Ala164His;Ala164Ile; Ala164Lys; Ala164Leu; Ala164Met; Ala164Asn; Ala164Pro;Ala164Gln; Ala164Arg; Ala164Ser; Ala164Thr; Ala164Val; Ala164Trp;Ala164Tyr; Leu165Ala; Leu165Cys; Leu165Asp; Leu165Glu; Leu165Phe;Leu165Gly; Leu165His; Leu165Ile; Leu165Lys; Leu165Met; Leu165Asn;Leu165Pro; Leu165Gln; Leu165Arg; Leu165Ser; Leu165Thr; Leu165Val;Leu165Trp; Leu165Tyr; Glu166Ala; Glu166Cys; Glu166Asp; Glu166Phe;Glu166Gly; Glu166His; Glu166Ile; Glu166Lys; Glu166Leu; Glu166Met;Glu166Asn; Glu166Pro; Glu166Gln; Glu166Arg; Glu166Ser; Glu166Thr;Glu166Val; Glu166Trp; Glu166Tyr; Asp167Ala; Asp167Cys; Asp167Glu;Asp167Phe; Asp167Gly; Asp167His; Asp167Ile; Asp167Lys; Asp167Leu;Asp167Met; Asp167Asn; Asp167Pro; Asp167Gln; Asp167Arg; Asp167Ser;Asp167Thr; Asp167Val; Asp167Trp; Asp167Tyr; Lys168Ala; Lys168Cys;Lys168Asp; Lys168Glu; Lys168Phe; Lys168Gly; Lys168His; Lys168Ile;Lys168Leu; Lys168Met; Lys168Asn; Lys168Pro; Lys168Gln; Lys168Arg;Lys168Ser; Lys168Thr; Lys168Val; Lys168Trp; Lys168Tyr; Leu169Ala;Leu169Cys; Leu169Asp; Leu169Glu; Leu169Phe; Leu169Gly; Leu169His;Leu169Ile; Leu169Lys; Leu169Met; Leu169Asn; Leu169Pro; Leu169Gln;Leu169Arg; Leu169Ser; Leu169Thr; Leu169Val; Leu169Trp; Leu169Tyr;Pro170Ala; Pro170Cys; Pro170Asp; Pro170Glu; Pro170Phe; Pro170Gly;Pro170His; Pro170Ile; Pro170Lys; Pro170Leu; Pro170Met; Pro170Asn;Pro170Gln; Pro170Arg; Pro170Ser; Pro170Thr; Pro170Val; Pro170Trp;Pro170Tyr; Ser171Ala; Ser171Cys; Ser171Asp; Ser171Glu; Ser171Phe;Ser171Gly; Ser171His; Ser171Ile; Ser171Lys; Ser171Leu; Ser171Met;Ser171Asn; Ser171Pro; Ser171Gln; Ser171Arg; Ser171Thr; Ser171Val;Ser171Trp; Ser171Tyr; Glu172Ala; Glu172Cys; Glu172Asp; Glu172Phe;Glu172Gly; Glu172His; Glu172Ile; Glu172Lys; Glu172Leu; Glu172Met;Glu172Asn; Glu172Pro; Glu172Gln; Glu172Arg; GluI72Ser; Glu172Thr;Glu172Val; Glu172Trp; Glu172Tyr; Lys173Ala; Lys173Cys; Lys173Asp;Lys173Glu; Lys173Phe; Lys173Gly; Lys173His; Lys173Ile; Lys173Leu;Lys173Met; Lys173Asn; Lys173Pro; Lys173Gln; Lys173Arg; Lys173Ser;Lys173Thr; Lys173Val; Lys173Trp; Lys173Tyr; Arg174Ala; Arg174Cys;Arg174Asp; Arg174Glu; Arg174Phe; Arg174Gly; Arg174His; Arg174Ile;Arg174Lys; Arg174Leu; Arg174Met; Arg174Asn; Arg174Pro; Arg174Gln;Arg174Ser; Arg174Thr; Arg174Val; Arg174Trp; Arg174Tyr; Glu175Ala;Glu175Cys; Glu175Asp; Glu175Phe; Glu175Gly; Glu175His; Glu175Ile;Glu175Lys; Glu175Leu; Glu175Met; Glu175Asn; Glu175Pro; Glu175Gln;Glu175Arg; Glu175Ser; Glu175Thr; Glu175Val; Glu175Trp; Glu175Tyr;Leu176Ala; Leu176Cys; Leu176Asp; Leu176Glu; Leu176Phe; Leu176Gly;Leu176His; Leu176Ile; Leu176Lys; Leu176Met; Leu176Asn; Leu176Pro;Leu176Gln; Leu176Arg; Leu176Ser; Leu176Thr; Leu176Val; Leu176Trp;Leu176Tyr; Leu177Ala; Leu177Cys; Leu177Asp; Leu177Glu; Leu177Phe;Leu177Gly; Leu177His; Leu177Ile; Leu177Lys; Leu177Met; Leu177Asn;Leu177Pro; Leu177Gln; Leu177Arg; Leu177Ser; Leu177Thr; Leu177Val;Leu177Trp; Leu177Tyr; Ile178Ala; IIe178Cys; IIe178Asp; IIe178Glu;IIe178Phe; IIe178Gly; IIe178His; IIe178Lys; IIe178Leu; IIe178Met;IIe178Asn; IIe178Pro; IIe178Gln; IIe178Arg; IIe178Ser; IIe178Thr;IIe178VaI; IIe178Trp; IIe178Tyr; Asp179Ala; Asp179Cys; Asp179Glu;Asp179Phe; Asp179Gly; Asp179His; Asp179Ile; Asp179Lys; Asp179Leu;Asp179Met; Asp179Asn; Asp179Pro; Asp179Gln; Asp179Arg; Asp179Ser;Asp179Thr; Asp179VaI; Asp179Trp; Asp179Tyr; Trp180Ala; Trp180Cys;Trp180Asp; Trp180Glu; Trp180Phe; Trp180Gly; Trp180His; Trp180Ile;Trp180Lys; Trp180Leu; Trp180Met; Trp180Asn; Trp180Pro; Trp180Gln;Trp180Arg; Trp180Ser; Trp180Thr; Trp180Val; Trp180Tyr; Met181Ala;Met181Cys; Met181Asp; Met181Glu; Met181Phe; Met181Gly; Met181His;Met181Ile; Met181Lys; Met181Leu; Met181Asn; Met181Pro; Met181Gln;Met181Arg; Met181Ser; Met181Thr; Met181Val; Met181Trp; Met181Tyr;Lys182Ala; Lys182Cys; Lys182Asp; Lys182Glu; Lys182Phe; Lys182Gly;Lys182His; Lys182Ile; Lys182Leu; Lys182Met; Lys182Asn; Lys182Pro;Lys182Gln; Lys182Arg; Lys182Ser; Lys182Thr; Lys182VaI; Lys182Trp;Lys182Tyr; Arg183Ala; Arg183Cys; Arg183Asp; Arg183Glu; Arg183Phe;Arg183Gly; Arg183His; Arg183Ile; Arg183Lys; Arg183Leu; Arg183Met;Arg183Asn; Arg183Pro; Arg183Gln; Arg183Ser; Arg183Thr; Arg183Val;Arg183Trp; Arg183Tyr; Asn184Ala; Asn184Cys; Asn184Asp; Asn184Glu;Asn184Phe; Asn184Gly; Asn184His; Asn184Ile; Asn184Lys; Asn184Leu;Asn184Met; Asn184Pro; Asn184Gln; Asn184Arg; Asn184Ser; Asn184Thr;Asn184VaI; Asn184Trp; Asn184Tyr; Thr185Ala; Thr185Cys; Thr185Asp;Thr185Glu; Thr185Phe; Thr185Gly; Thr185His; Thr185Ile; Thr185Lys;Thr185Leu; Thr185Met; Thr185Asn; Thr185Pro; Thr185Gln; Thr185Arg;Thr185Ser; Thr185VaI; Thr185Trp; Thr185Tyr; Thr186Ala; Thr186Cys;Thr186Asp; Thr186Glu; Thr186Phe; Thr186Gly; Thr186His; Thr186Ile;Thr186Lys; Thr186Leu; Thr186Met; Thr186Asn; Thr186Pro; Thr186Gln;Thr186Arg; Thr186Ser; Thr186VaI; Thr186Trp; Thr186Tyr; Gly187Ala;Gly187Cys; Gly187Asp; Gly187Glu; Gly187Phe; Gly187His; Gly187IIe;Gly187Lys; Gly187Leu; Gly187Met; Gly187Asn; Gly187Pro; Gly187Gln;Gly187Arg; Gly187Ser; Gly187Thr; Gly187VaI; Gly187Trp; Gly187Tyr;Asp188Ala; Asp188Cys; Asp188Glu; Asp188Phe; Asp188Gly; Asp188His;Asp188Ile; Asp188Lys; Asp188Leu; Asp188Met; Asp188Asn; Asp188Pro;Asp188Gln; Asp188Arg; Asp188Ser; Asp188Thr; Asp188Val; Asp188Trp;Asp188Tyr; Ala189Cys; Ala189Asp; Ala189Glu; Ala189Phe; Ala189Gly;Ala189His; Ala189IIe; Ala189Lys; Ala189Leu; Ala189Met; Ala189Asn;Ala189Pro; Ala189Gln; Ala189Arg; Ala189Ser; Ala189Thr; Ala189VaI;Ala189Trp; Ala189Tyr; Leu190Ala; Leu190Cys; Leu190Asp; Leu190Glu;Leu190Phe; Leu190Gly; Leu190His; Leu190Ile; Leu190Lys; Leu190Met;Leu190Asn; Leu190Pro; Leu190Gln; Leu190Arg; Leu190Ser; Leu190Thr;Leu190Val; Leu190Trp; Leu190Tyr; IIe191Ala; IIe191Cys; IIe191Asp;IIe191Glu; IIe191Phe; IIe191Gly; IIe191His; IIe191Lys; IIe191Leu;IIe191Met; IIe191Asn; IIe191Pro; IIe191Gln; IIe191Arg; IIe191Ser;IIe191Thr; IIe191VaI; IIe191Trp; IIe191Tyr; Arg192Ala; Arg192Cys;Arg192Asp; Arg192Glu; Arg192Phe; Arg192Gly; Arg192His; Arg192Ile;Arg192Lys; Arg192Leu; Arg192Met; Arg192Asn; Arg192Pro; Arg192Gln;Arg192Ser; Arg192Thr; Arg192Val; Arg192Trp; Arg192Tyr; Ala193Cys;Ala193Asp; Ala193Glu; Ala193Phe; Ala193Gly; Ala193His; Ala193IIe;Ala193Lys; Ala193Leu; Ala193Met; Ala193Asn; Ala193Pro; Ala193Gln;Ala193Arg; Ala193Ser; Ala193Thr; Ala193VaI; Ala193Trp; Ala193Tyr;Gly194Ala; Gly194Cys; Gly194Asp; Gly194Glu; Gly194Phe; Gly194His;Gly194Ile; Gly194Lys; Gly194Leu; Gly194Met; Gly194Asn; Gly194Pro;Gly194Gln; Gly194Arg; Gly194Ser; Gly194Thr; Gly194Val; Gly194Trp;Gly194Tyr; Val195Ala; Val195Cys; Val195Asp; Val195Glu; Val195Phe;Val195Gly; Val195His; Val195Ile; Val195Lys; Val195Leu; Val195Met;Val195Asn; Val195Pro; Val195Gln; Val195Arg; Val195Ser; Val195Thr;Val195Trp; Val195Tyr; Pro196Ala; Pro196Cys; Pro196Asp; Pro196Glu;Pro196Phe; Pro196Gly; Pro196His; Pro196Ile; Pro196Lys; Pro196Leu;Pro196Met; Pro196Asn; Pro196Gln; Pro196Arg; Pro196Ser; Pro196Thr;Pro196Val; Pro196Trp; Pro196Tyr; Asp197Ala; Asp197Cys; Asp197Glu;Asp197Phe; Asp197Gly; Asp197His; Asp197Ile; Asp197Lys; Asp197Leu;Asp197Met; Asp197Asn; Asp197Pro; Asp197Gln; Asp197Arg; Asp197Ser;Asp197Thr; Asp197Val; Asp197Trp; Asp197Tyr; Gly198Ala; Gly198Cys;Gly198Asp; Gly198Glu; Gly198Phe; Gly198His; Gly198Ile; Gly198Lys;Gly198Leu; Gly198Met; Gly198Asn; Gly198Pro; Gly198Gln; Gly198Arg;Gly198Ser; Gly198Thr; Gly198Val; Gly198Trp; Gly198Tyr; Trp199Ala;Trp199Cys; Trp199Asp; Trp199Glu; Trp199Phe; Trp199Gly; Trp199His;Trp199Ile; Trp199Lys; Trp199Leu; Trp199Met; Trp199Asn; Trp199Pro;Trp199Gln; Trp199Arg; Trp199Ser; Trp199Thr; Trp199Val; Trp199Tyr;Glu200Ala; Glu200Cys; Glu200Asp; Glu200Phe; Glu200Gly; Glu200His;Glu200Ile; Glu200Lys; Glu200Leu; Glu200Met; Glu200Asn; Glu200Pro;Glu200Gln; Glu200Arg; Glu200Ser; Glu200Thr; Glu200Val; Glu200Trp;Glu200Tyr; Val201Ala; Val201Cys; Val201Asp; Val201Glu; Val201Phe;Val201Gly; Val201His; Val201Ile; Val201Lys; Val201Leu; Val201Met;Val201Asn; Val201Pro; Val201Gln; Val201Arg; Val201Ser; Val201Thr;Val201Trp; Val201Tyr; Ala202Cys; Ala202Asp; Ala202Glu; Ala202Phe;Ala202Gly; Ala202His; Ala202Ile; Ala202Lys; Ala202Leu; Ala202Met;Ala202Asn; Ala202Pro; Ala202Gln; Ala202Arg; Ala202Ser; Ala202Thr;Ala202Val; Ala202Trp; Ala202Tyr; Asp203Ala; Asp203Cys; Asp203Glu;Asp203Phe; Asp203Gly; Asp203His; Asp203Ile; Asp203Lys; Asp203Leu;Asp203Met; Asp203Asn; Asp203Pro; Asp203Gln; Asp203Arg; Asp203Ser;Asp203Thr; Asp203Val; Asp203Trp; Asp203Tyr; Lys204Ala; Lys204Cys;Lys204Asp; Lys204Glu; Lys204Phe; Lys204Gly; Lys204His; Lys204Ile;Lys204Leu; Lys204Met; Lys204Asn; Lys204Pro; Lys204Gln; Lys204Arg;Lys204Ser; Lys204Thr; Lys204Val; Lys204Trp; Lys204Tyr; Thr205Ala;Thr205Cys; Thr205Asp; Thr205Glu; Thr205Phe; Thr205Gly; Thr205His;Thr205Ile; Thr205Lys; Thr205Leu; Thr205Met; Thr205Asn; Thr205Pro;Thr205Gln; Thr205Arg; Thr205Ser; Thr205Val; Thr205Trp; Thr205Tyr;Gly206Ala; Gly206Cys; Gly206Asp; Gly206Glu; Gly206Phe; Gly206His;Gly206Ile; Gly206Lys; Gly206Leu; Gly206Met; Gly206Asn; Gly206Pro;Gly206Gln; Gly206Arg; Gly206Ser; Gly206Thr; Gly206Val; Gly206Trp;Gly206Tyr; Ala207Cys; Ala207Asp; Ala207Glu; Ala207Phe; Ala207Gly;Ala207His; Ala207Ile; Ala207Lys; Ala207Leu; Ala207Met; Ala207Asn;Ala207Pro; Ala207Gln; Ala207Arg; Ala207Ser; Ala207Thr; Ala207Val;Ala207Trp; Ala207Tyr; Ala208Cys; Ala208Asp; Ala208Glu; Ala208Phe;Ala208Gly; Ala208His; Ala208Ile; Ala208Lys; Ala208Leu; Ala208Met;Ala208Asn; Ala208Pro; Ala208Gln; Ala208Arg; Ala208Ser; Ala208Thr;Ala208Val; Ala208Trp; Ala208Tyr; Ser209Ala; Ser209Cys; Ser209Asp;Ser209Glu; Ser209Phe; Ser209Gly; Ser209His; Ser209Ile; Ser209Lys;Ser209Leu; Ser209Met; Ser209Asn; Ser209Pro; Ser209Gln; Ser209Arg;Ser209Thr; Ser209Val; Ser209Trp; Ser209Tyr; Tyr210Ala; Tyr210Cys;Tyr210Asp; Tyr210Glu; Tyr210Phe; Tyr210Gly; Tyr210H is; Tyr210Ile;Tyr210Lys; Tyr210Leu; Tyr210Met; Tyr210Asn; Tyr210Pro; Tyr210Gln;Tyr210Arg; Tyr210Ser; Tyr210Thr; Tyr210Val; Tyr210Trp; Gly211Ala;Gly211Cys; Gly211Asp; Gly211Glu; Gly211Phe; Gly211His; Gly211Ile;Gly211Lys; Gly211Leu; Gly211Met; Gly211Asn; Gly211Pro; Gly211Gln;Gly211Arg; Gly211Ser; Gly211Thr; Gly211Val; Gly211Trp; Gly211Tyr;Thr212Ala; Thr212Cys; Thr212Asp; Thr212Glu; Thr212Phe; Thr212Gly;Thr212His; Thr212Ile; Thr212Lys; Thr212Leu; Thr212Met; Thr212Asn;Thr212Pro; Thr212Gln; Thr212Arg; Thr212Ser; Thr212Val; Thr212Trp;Thr212Tyr; Arg213Ala; Arg213Cys; Arg213Asp; Arg213Glu; Arg213Phe;Arg213Gly; Arg213His; Arg213Ile; Arg213Lys; Arg213Leu; Arg213Met;Arg213Asn; Arg213Pro; Arg213Gln; Arg213Ser; Arg213Thr; Arg213Val;Arg213Trp; Arg213Tyr; Asn214Ala; Asn214Cys; Asn214Asp; Asn214Glu;Asn214Phe; Asn214Gly; Asn214His; Asn214Ile; Asn214Lys; Asn214Leu;Asn214Met; Asn214Pro; Asn214Gln; Asn214Arg; Asn214Ser; Asn214Thr;Asn214Val; Asn214Trp; Asn214Tyr; Asp215Ala; Asp215Cys; Asp215Glu;Asp215Phe; Asp215Gly; Asp215His; Asp215Ile; Asp215Lys; Asp215Leu;Asp215Met; Asp215Asn; Asp215Pro; Asp215Gln; Asp215Arg; Asp215Ser;Asp215Thr; Asp215Val; Asp215Trp; Asp215Tyr; Ile216Ala; Ile216Cys;Ile216Asp; Ile216Glu; Ile216Phe; Ile216Gly; Ile216His; Ile216Lys;Ile216Leu; Ile216Met; Ile216Asn; Ile216Pro; Ile216Gln; Ile216Arg;Ile216Ser; Ile216Thr; Ile216Val; Ile216Trp; Ile216Tyr; Ala217Cys;Ala217Asp; Ala217Glu; Ala217Phe; Ala217Gly; Ala217His; Ala217Ile;Ala217Lys; Ala217Leu; Ala217Met; Ala217Asn; Ala217Pro; Ala217Gln;Ala217Arg; Ala217Ser; Ala217Thr; Ala217Val; Ala217Trp; Ala217Tyr;Ile218Ala; Ile218Cys; Ile218Asp; Ile218Glu; Ile218Phe; Ile218Gly;Ile218His; Ile218Lys; Ile218Leu; Ile218Met; Ile218Asn; Ile218Pro;Ile218Gln; Ile218Arg; Ile218Ser; Ile218Thr; Ile218Val; Ile218Trp;Ile218Tyr; Ile219Ala; Ile219Cys; Ile219Asp; Ile219Glu; Ile219Phe;Ile219Gly; Ile219His; Ile219Lys; Ile219Leu; Ile219Met; Ile219Asn;Ile219Pro; Ile219Gln; Ile219Arg; Ile219Ser; Ile219Thr; Ile219Val;Ile219Trp; Ile219Tyr; Trp220Ala; Trp220Cys; Trp220Asp; Trp220Glu;Trp220Phe; Trp220Gly; Trp220His; Trp220Ile; Trp220Lys; Trp220Leu;Trp220Met; Trp220Asn; Trp220Pro; Trp220Gln; Trp220Arg; Trp220Ser;Trp220Thr; Trp220Val; Trp220Tyr; Pro221Ala; Pro221Cys; Pro221Asp;Pro221Glu; Pro221Phe; Pro221Gly; Pro221His; Pro221Ile; Pro221Lys;Pro221Leu; Pro221Met; Pro221Asn; Pro221Gln; Pro221Arg; Pro221Ser;Pro221Thr; Pro221Val; Pro221Trp; Pro221Tyr; Pro222Ala; Pro222Cys;Pro222Asp; Pro222Glu; Pro222Phe; Pro222Gly; Pro222His; Pro222Ile;Pro222Lys; Pro222Leu; Pro222Met; Pro222Asn; Pro222Gln; Pro222Arg;Pro222Ser; Pro222Thr; Pro222Val; Pro222Trp; Pro222Tyr; Lys223Ala;Lys223Cys; Lys223Asp; Lys223Glu; Lys223Phe; Lys223Gly; Lys223His;Lys223Ile; Lys223Leu; Lys223Met; Lys223Asn; Lys223Pro; Lys223Gln;Lys223Arg; Lys223Ser; Lys223Thr; Lys223Val; Lys223Trp; Lys223Tyr;Gly224Ala; Gly224Cys; Gly224Asp; Gly224Glu; Gly224Phe; Gly224His;Gly224Ile; Gly224Lys; Gly224Leu; Gly224Met; Gly224Asn; Gly224Pro;Gly224Gln; Gly224Arg; Gly224Ser; Gly224Thr; Gly224Val; Gly224Trp;Gly224Tyr; Asp225Ala; Asp225Cys; Asp225Glu; Asp225Phe; Asp225Gly;Asp225His; Asp225Ile; Asp225Lys; Asp225Leu; Asp225Met; Asp225Asn;Asp225Pro; Asp225Gln; Asp225Arg; Asp225Ser; Asp225Thr; Asp225Val;Asp225Trp; Asp225Tyr; Pro226Ala; Pro226Cys; Pro226Asp; Pro226Glu;Pro226Phe; Pro226Gly; Pro226His; Pro226Ile; Pro226Lys; Pro226Leu;Pro226Met; Pro226Asn; Pro226Gln; Pro226Arg; Pro226Ser; Pro226Thr;Pro226Val; Pro226Trp; Pro226Tyr; Val227Ala; Val227Cys; Val227Asp;Val227Glu; Val227Phe; Val227Gly; Val227His; Val227Ile; Val227Lys;Val227Leu; Val227Met; Val227Asn; Val227Pro; Val227Gln; Val227Arg;Val227Ser; Val227Thr; Val227Trp; Val227Tyr; Val228Ala; Val228Cys;Val228Asp; Val228Glu; Val228Phe; Val228Gly; Val228His; Val228Ile;Val228Lys; Val228Leu; Val228Met; Val228Asn; Val228Pro; Val228Gln;Val228Arg; Val228Ser; Val228Thr; Val228Trp; Val228Tyr; Leu229Ala;Leu229Cys; Leu229Asp; Leu229Glu; Leu229Phe; Leu229Gly; Leu229His;Leu229Ile; Leu229Lys; Leu229Met; Leu229Asn; Leu229Pro; Leu229Gln;Leu229Arg; Leu229Ser; Leu229Thr; Leu229Val; Leu229Trp; Leu229Tyr;Ala230Cys; Ala230Asp; Ala230Glu; Ala230Phe; Ala230Gly; Ala230His;Ala230Ile; Ala230Lys; Ala230Leu; Ala230Met; Ala230Asn; Ala230Pro;Ala230Gln; Ala230Arg; Ala230Ser; Ala230Thr; Ala230Val; Ala230Trp;Ala230Tyr; Val231Ala; Val231Cys; Val231Asp; Val231Glu; Val231Phe;Val231Gly; Val231His; Val231Ile; Val231Lys; Val231Leu; Val231Met;Val231Asn; Val231Pro; Val231Gln; Val231Arg; Val231Ser; Val231Thr;Val231Trp; Val231Tyr; Leu232Ala; Leu232Cys; Leu232Asp; Leu232Glu;Leu232Phe; Leu232Gly; Leu232His; Leu232Ile; Leu232Lys; Leu232Met;Leu232Asn; Leu232Pro; Leu232Gln; Leu232Arg; Leu232Ser; Leu232Thr;Leu232Val; Leu232Trp; Leu232Tyr; Ser233Ala; Ser233Cys; Ser233Asp;Ser233Glu; Ser233Phe; Ser233Gly; Ser233His; Ser233Ile; Ser233Lys;Ser233Leu; Ser233Met; Ser233Asn; Ser233Pro; Ser233Gln; Ser233Arg;Ser233Thr; Ser233Val; Ser233Trp; Ser233Tyr; Ser234Ala; Ser234Cys;Ser234Asp; Ser234Glu; Ser234Phe; Ser234Gly; Ser234His; Ser234Ile;Ser234Lys; Ser234Leu; Ser234Met; Ser234Asn; Ser234Pro; Ser234Gln;Ser234Arg; Ser234Thr; Ser234Val; Ser234Trp; Ser234Tyr; Arg235Ala;Arg235Cys; Arg235Asp; Arg235Glu; Arg235Phe; Arg235Gly; Arg235His;Arg235Ile; Arg235Lys; Arg235Leu; Arg235Met; Arg235Asn; Arg235Pro;Arg235Gln; Arg235Ser; Arg235Thr; Arg235Val; Arg235Trp; Arg235Tyr;Asp236Ala; Asp236Cys; Asp236Glu; Asp236Phe; Asp236Gly; Asp236His;Asp236Ile; Asp236Lys; Asp236Leu; Asp236Met; Asp236Asn; Asp236Pro;Asp236Gln; Asp236Arg; Asp236Ser; Asp236Thr; Asp236Val; Asp236Trp;Asp236Tyr; Lys237Ala; Lys237Cys; Lys237Asp; Lys237Glu; Lys237Phe;Lys237Gly; Lys237His; Lys237Ile; Lys237Leu; Lys237Met; Lys237Asn;Lys237Pro; Lys237Gln; Lys237Arg; Lys237Ser; Lys237Thr; Lys237Val;Lys237Trp; Lys237Tyr; Lys238Ala; Lys238Cys; Lys238Asp; Lys238Glu;Lys238Phe; Lys238Gly; Lys238His; Lys238Ile; Lys238Leu; Lys238Met;Lys238Asn; Lys238Pro; Lys238Gln; Lys238Arg; Lys238Ser; Lys238Thr;Lys238Val; Lys238Trp; Lys238Tyr; Asp239Ala; Asp239Cys; Asp239Glu;Asp239Phe; Asp239Gly; Asp239His; Asp239Ile; Asp239Lys; Asp239Leu;Asp239Met; Asp239Asn; Asp239Pro; Asp239Gln; Asp239Arg; Asp239Ser;Asp239Thr; Asp239Val; Asp239Trp; Asp239Tyr; Ala240Cys; Ala240Asp;Ala240Glu; Ala240Phe; Ala240Gly; Ala240His; Ala240Ile; Ala240Lys;Ala240Leu; Ala240Met; Ala240Asn; Ala240Pro; Ala240Gln; Ala240Arg;Ala240Ser; Ala240Thr; Ala240Val; Ala240Trp; Ala240Tyr; Lys241Ala;Lys241Cys; Lys241Asp; Lys241Glu; Lys241Phe; Lys241Gly; Lys241His;Lys241Ile; Lys241Leu; Lys241Met; Lys241Asn; Lys241Pro; Lys241Gln;Lys241Arg; Lys241Ser; Lys241Thr; Lys241Val; Lys241Trp; Lys241Tyr;Tyr242Ala; Tyr242Cys; Tyr242Asp; Tyr242Glu; Tyr242Phe; Tyr242Gly;Tyr242His; Tyr242Ile; Tyr242Lys; Tyr242Leu; Tyr242Met; Tyr242Asn;Tyr242Pro; Tyr242Gln; Tyr242Arg; Tyr242Ser; Tyr242Thr; Tyr242Val;Tyr242Trp; Asp243Ala; Asp243Cys; Asp243Glu; Asp243Phe; Asp243Gly;Asp243His; Asp243Ile; Asp243Lys; Asp243Leu; Asp243Met; Asp243Asn;Asp243Pro; Asp243Gln; Asp243Arg; Asp243Ser; Asp243Thr; Asp243Val;Asp243Trp; Asp243Tyr; Asp244Ala; Asp244Cys; Asp244Glu; Asp244Phe;Asp244Gly; Asp244His; Asp244Ile; Asp244Lys; Asp244Leu; Asp244Met;Asp244Asn; Asp244Pro; Asp244Gln; Asp244Arg; Asp244Ser; Asp244Thr;Asp244Val; Asp244Trp; Asp244Tyr; Lys245Ala; Lys245Cys; Lys245Asp;Lys245Glu; Lys245Phe; Lys245Gly; Lys245His; Lys245Ile; Lys245Leu;Lys245Met; Lys245Asn; Lys245Pro; Lys245Gln; Lys245Arg; Lys245Ser;Lys245Thr; Lys245Val; Lys245Trp; Lys245Tyr; Leu246Ala; Leu246Cys;Leu246Asp; Leu246Glu; Leu246Phe; Leu246Gly; Leu246His; Leu246Ile;Leu246Lys; Leu246Met; Leu246Asn; Leu246Pro; Leu246Gln; Leu246Arg;Leu246Ser; Leu246Thr; Leu246Val; Leu246Trp; Leu246Tyr; Ile247Ala;Ile247Cys; Ile247Asp; Ile247Glu; Ile247Phe; Ile247Gly; Ile247His;Ile247Lys; Ile247Leu; Ile247Met; Ile247Asn; Ile247Pro; Ile247Gln;Ile247Arg; Ile247Ser; Ile247Thr; Ile247Val; Ile247Trp; Ile247Tyr;Ala248Cys; Ala248Asp; Ala248Glu; Ala248Phe; Ala248Gly; Ala248His;Ala248Ile; Ala248Lys; Ala248Leu; Ala248Met; Ala248Asn; Ala248Pro;Ala248Gln; Ala248Arg; Ala248Ser; Ala248Thr; Ala248Val; Ala248Trp;Ala248Tyr; Glu249Ala; Glu249Cys; Glu249Asp; Glu249Phe; Glu249Gly;Glu249His; Glu249Ile; Glu249Lys; Glu249Leu; Glu249Met; Glu249Asn;Glu249Pro; Glu249Gln; Glu249Arg; Glu249Ser; Glu249Thr; Glu249Val;Glu249Trp; Glu249Tyr; Ala250Cys; Ala250Asp; Ala250Glu; Ala250Phe;Ala250Gly; Ala250His; Ala250Ile; Ala250Lys; Ala250Leu; Ala250Met;Ala250Asn; Ala250Pro; Ala250Gln; Ala250Arg; Ala250Ser; Ala250Thr;Ala250Val; Ala250Trp; Ala250Tyr; Thr251Ala; Thr251Cys; Thr251Asp;Thr251Glu; Thr251Phe; Thr251Gly; Thr251His; Thr251Ile; Thr251Lys;Thr251Leu; Thr251Met; Thr251Asn; Thr251Pro; Thr251Gln; Thr251Arg;Thr251Ser; Thr251Val; Thr251Trp; Thr251Tyr; Lys252Ala; Lys252Cys;Lys252Asp; Lys252Glu; Lys252Phe; Lys252Gly; Lys252His; Lys252Ile;Lys252Leu; Lys252Met; Lys252Asn; Lys252Pro; Lys252Gln; Lys252Arg;Lys252Ser; Lys252Thr; Lys252Val; Lys252Trp; Lys252Tyr; Val253Ala;Val253Cys; Val253Asp; Val253Glu; Val253Phe; Val253Gly; Val253His;Val253Ile; Val253Lys; Val253Leu; Val253Met; Val253Asn; Val253Pro;Val253Gln; Val253Arg; Val253Ser; Val253Thr; Val253Trp; Val253Tyr;Val254Ala; Val254Cys; Val254Asp; Val254Glu; Val254Phe; Val254Gly;Val254His; Val254Ile; Val254Lys; Val254Leu; Val254Met; Val254Asn;Val254Pro; Val254Gln; Val254Arg; Val254Ser; Val254Thr; Val254Trp;Val254Tyr; Met255Ala; Met255Cys; Met255Asp; Met255Glu; Met255Phe;Met255Gly; Met255His; Met255Ile; Met255Lys; Met255Leu; Met255Asn;Met255Pro; Met255Gln; Met255Arg; Met255Ser; Met255Thr; Met255Val;Met255Trp; Met255Tyr; Lys256Ala; Lys256Cys; Lys256Asp; Lys256Glu;Lys256Phe; Lys256Gly; Lys256His; Lys256Ile; Lys256Leu; Lys256Met;Lys256Asn; Lys256Pro; Lys256Gln; Lys256Arg; Lys256Ser; Lys256Thr;Lys256Val; Lys256Trp; Lys256Tyr; Ala257Cys; Ala257Asp; Ala257Glu;Ala257Phe; Ala257Gly; Ala257His; Ala257Ile; Ala257Lys; Ala257Leu;Ala257Met; Ala257Asn; Ala257Pro; Ala257Gln; Ala257Arg; Ala257Ser;Ala257Thr; Ala257Val; Ala257Trp; Ala257Tyr; Leu258Ala; Leu258Cys;Leu258Asp; Leu258Glu; Leu258Phe; Leu258Gly; Leu258His; Leu258Ile;Leu258Lys; Leu258Met; Leu258Asn; Leu258Pro; Leu258Gln; Leu258Arg;Leu258Ser; Leu258Thr; Leu258Val; Leu258Trp; Leu258Tyr; Asn259Ala;Asn259Cys; Asn259Asp; Asn259Glu; Asn259Phe; Asn259Gly; Asn259His;Asn259Ile; Asn259Lys; Asn259Leu; Asn259Met; Asn259Pro; Asn259Gln;Asn259Arg; Asn259Ser; Asn259Thr; Asn259Val; Asn259Trp; Asn259Tyr;Met260Ala; Met260Cys; Met260Asp; Met260Glu; Met260Phe; Met260Gly;Met260His; Met260Ile; Met260Lys; Met260Leu; Met260Asn; Met260Pro;Met260Gln; Met260Arg; Met260Ser; Met260Thr; Met260Val; Met260Trp;Met260Tyr; Asn261Ala; Asn261Cys; Asn261Asp; Asn261Glu; Asn261Phe;Asn261Gly; Asn261His; Asn261Ile; Asn261Lys; Asn261Leu; Asn261Met;Asn261Pro; Asn261Gln; Asn261Arg; Asn261Ser; Asn261Thr; Asn261Val;Asn261Trp; Asn261Tyr; Gly262Ala; Gly262Cys; Gly262Asp; Gly262Glu;Gly262Phe; Gly262His; Gly262Ile; Gly262Lys; Gly262Leu; Gly262Met;Gly262Asn; Gly262Pro; Gly262Gln; Gly262Arg; Gly262Ser; Gly262Thr;Gly262Val; Gly262Trp; Gly262Tyr; Lys263Ala; Lys263Cys; Lys263Asp;Lys263Glu; Lys263Phe; Lys263Gly; Lys263His; Lys263Ile; Lys263Leu;Lys263Met; Lys263Asn; Lys263Pro; Lys263Gln; Lys263Arg; Lys263Ser;Lys263Thr; Lys263Val; Lys263Trp; Lys263Tyr; Met 264Ala; Met 264Cys; Met264Asp; Met 264Glu; Met 264Phe; Met 264Gly; Met 264His; Met 264Ile; Met264Lys; Met 264Leu; Met 264Asn; Met 264Pro; Met 264Gln; Met 264Arg; Met264Ser; Met 264Thr; Met 264Val; Met 264Trp; Met 264Tyr; Asn 265Ala; Asn265Cys; Asn 265Asp; Asn 265Glu; Asn 265Phe; Asn 265Gly; Asn 265His; Asn265Ile; Asn 265Lys; Asn 265Leu; Asn 265Met; Asn 265Pro; Asn 265Gln; Asn265Arg; Asn 265Ser; Asn 265Thr; Asn 265Val; Asn 265Trp; Asn 265Tyr; Gly266Ala; Gly 266Cys; Gly 266Asp; Gly 266Glu; Gly 266Phe; Gly 266His; Gly266Ile; Gly 266Lys; Gly 266Leu; Gly 266Met; Gly 266Asn; Gly 266Pro; Gly266Gln; Gly 266Arg; Gly 266Ser; Gly 266Thr; Gly 266Val; Gly 266Trp; Gly266Tyr; Lys267Ala; Lys267Cys; Lys267Asp; Lys267Glu; Lys267Phe;Lys267Gly; Lys267His; Lys267Ile; Lys267Leu; Lys267Met; Lys267Asn;Lys267Pro; Lys267Gln; Lys267Arg; Lys267Ser; Lys267Thr; Lys267Val;Lys267Trp; and Lys267Tyr. In some embodiments, SEQ ID NO: 1 may have aMet and/or Thr preceeding the first residue of the sequence. Theseresidues may be similarly mutated as above.

In all of these mutants, the numbering of residues corresponds to SEQ IDNO: 1. These residue numbers may be converted to Ambler numbers (Ambleret al., 1991, A standard numbering scheme for the Class A β-lactamases,Biochem. J. 276:269-272, the contents of which are hereby incorporatedby reference) through use of any conventional bioinformatic method, forexample by using BLAST (Basic Local Alignment Search Tools) or FASTA(FAST-All). For example, residue 244 corresponds to Ambler 276. Forexample, the following conversions may be used:

Ambler SEQ ID NO: 1 Classification No. Residue F33 F6 I72 I44 Q135 Q105G156 G126 T160 T130 A232 A202 A237 A207 A238 A208 S240 S209 T243 T212R244 R213 S266 S234 D276 D244

Furthermore, percent identity may also be assessed with theseconventional bioinformatic methods.

In one aspect, the present invention pertains to beta-lactamases and/orpharmaceutical compositions comprising an amino acid sequence having atleast 70% (e.g. about 70%, or about 71%, or about 72%, or about 73%, orabout 74%, or about 75%, or about 76%, or about 77%, or about 78%, orabout 79%, or about 80%, or about 81%, or about 82%, or about 83%, orabout 84%, or about 85%, or about 86%, or about 87%, or about 88%, orabout 89%, or about 90%, or about 91%, or about 92%, or about 93%, orabout 94%, or about 95%, or about 96%, or about 97%, or about 98%, orabout 99%) sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3 and oneor more of the following mutations of Ambler classification: F33X,Q135X, G156X, A232X, A237X, A238X, S240X, 1243X, R244X, S266X, andD276X, wherein X is any naturally-occurring amino acid and with theproviso that D276X is not present in the context of a single mutant. Insome embodiments, X is a naturally occurring hydrophilic or hydrophobicamino acid residue or a non-classical amino acid.

In another aspect, the present invention pertains to beta-lactamasesand/or pharmaceutical compositions comprising an amino acid sequencehaving at least 70% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3and one or more of the following mutations of Ambler classification: ahydrophobic residue other than phenylalanine (F) at position 33; ahydrophobic residue other than glutamine (Q) at position 135; ahydrophilic residue other than glycine (G) at position 156; ahydrophobic residue other than alanine (A) at position 232; ahydrophilic residue other than alanine (A) at position 237; ahydrophobic or hydrophilic residue other than alanine (A) at position238; a hydrophilic residue other than serine (S) at position 240; ahydrophobic residue other than threonine (T) at position 243; ahydrophobic residue other than arginine (R) at position 244; ahydrophilic residue other than serine (S) at position 266; and ahydrophilic residue other than aspartate (D) at position 276, with theproviso that hydrophilic amino acid residue other than aspartic acid (D)at a position corresponding to position 276 is not present in thecontext of a single mutant.

As used throughout, a hydrophilic amino acid residue may include a polarand positively charged hydrophilic residue selected from arginine (R)and lysine (K), a polar and neutral of charge hydrophilic residueselected from asparagine (N), glutamine (Q), serine (S), threonine (T),proline (P), and cysteine (C), a polar and negatively chargedhydrophilic residue selected from aspartate (D) and glutamate (E), or anaromatic, polar and positively charged hydrophilic including histidine(H). As used throughout, a hydrophobic amino acid residue may include ahydrophobic, aliphatic amino acid selected from glycine (G), alanine(A), leucine (L), isoleucine (I), methionine (M), or valine (V) or ahydrophobic, aromatic amino acid selected from phenylalanine (F),tryptophan (W), or tyrosine (Y).

Mutations may be made to the gene sequence of a beta-lactamase (e.g. SEQID NOs: 3 and 4) by reference to the genetic code, including taking intoaccount codon degeneracy.

In some embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise one or more of the following mutations atpositions of Ambler classification: F33Y, Q135M, G156R, A232G, A237S,A238G or T, S240P or D, 12431, R244T, S266N, D276N or R or K, providedthat D276N or R or K is not in the context of a single mutant. In oneembodiment, the beta-lactamases and/or pharmaceutical compositionscomprise Q135M. In another embodiment, the beta-lactamases and/orpharmaceutical compositions comprise G156R and A238T. In anotherembodiment, the beta-lactamases and/or pharmaceutical compositionscomprise F33Y and D276N. In still another embodiment, thebeta-lactamases and/or pharmaceutical compositions comprise F33Y, S240P,and D276N. In one embodiment, the beta-lactamases and/or pharmaceuticalcompositions comprise F33Y, A238T, and D276N. In another embodiment, thebeta-lactamases and/or pharmaceutical compositions comprise A232G,A237S, A238G, and S240D. In a further embodiment, the beta-lactamasesand/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D,and R2441. In another embodiment, the beta-lactamases and/orpharmaceutical compositions comprise A232G, A237S, A238G, S240D, andD276R. In one embodiment, the beta-lactamases and/or pharmaceuticalcompositions comprise A232G, A237S, A238G, S240D, and D276K. In oneembodiment, the beta-lactamases and/or pharmaceutical compositionscomprise A232G, A237S, A238G, S240D, and Q135M. In one embodiment, thebeta-lactamases and/or pharmaceutical compositions comprise A238T. Inone embodiment, the beta-lactamases and/or pharmaceutical compositionscomprise 12431, S266N, and D276N. In one embodiment, the beta-lactamasesand/or pharmaceutical compositions comprise A232G, A237S, A238G, S240D,and D276N.

In other embodiments, the beta-lactamases and/or pharmaceuticalcompositions comprise an amino acid sequence having at least 70%sequence identity with SEQ ID NO: 2 and the following of Amblerclassification: a hydrophobic residue other than alanine (A) at position232; a hydrophilic residue other than alanine (A) at position 237; ahydrophobic residue other than alanine (A) at position 238; ahydrophilic residue other than serine (S) at position 240; and ahydrophilic residue other than aspartate (D) at position 276. In someembodiments, the hydrophobic residue other than alanine (A) at position232 is glycine (G). In some embodiments, the hydrophilic residue otherthan alanine (A) at position 237 is serine (S). In some embodiments, thehydrophobic residue other than alanine (A) at position 238 is glycine(G). In some embodiments, the hydrophilic residue other than serine (S)at position 240 is aspartate (D). In some embodiments, the other thanaspartate (D) at position 276 is asparagine (N). In some embodiments,the beta-lactamase and/or pharmaceutical composition comprises one ormore of A232G, A2375, A238G, S240D, and D276N. In some embodiments, thebeta-lactamase and/or pharmaceutical composition comprises all of A232G,A237S, A238G, S240D, and D276N, the sequence of which may be SEQ IDNO:5:

SEQ ID NO: 5 EMKDDFAKLEEQFDAKLGIFALDTGTNRTVAYRPDERFAFASTIKALTVGVLLQQKSIEDLNQRITTRDDLVNYNPITEKHVDTGMTLKELADASLRYSDNAAQNLILKQIGGPESLKKELRKIGDEVTNPERFEPELNEVNPGETQDTSTARALVTSLRAFALEDKLPSEKRELLIDWMKRNTTGDALIRAGVPDGWEVGDKTGSGDYGTRNDIAIIWPPKGDPVVLAVLSSRDKKDAKYDNKLIAEAT KVVMKALNMNGK orSEQ ID NO: 6:SEQ ID NO: 5, including the signal and the addition of the QASKT amino acids (the coding region if underlined):M I Q K R K R T V S F R L V L M C T L L F V S L P I T K T SA Q A S K T E M K D D F A K L E E Q F D A K L G I F A L D TG T N R T V A Y R P D E R F A F A S T I K A L T V G V L L QQ K S I E D L N Q R I T Y T R D D L V N Y N P I T E K H V DT G M T L K E L A D A S L R Y S D N A A Q N L I L K Q I G GP E S L K K E L R K I G D E V T N P E R F E P E L N E V N PG E T Q D T S T A R A L V T S L R A F A L E D K L P S E K RE L L I D W M K R N T T G D A L I R A G V P D G V V E V G DK T G S G D Y G T R N D I A I I W P P K G D P V V L A V L SS R D K K D A K Y D N K L I A E A T K V V M K A L N M N G K

The invention also provides for polynucleotides encoding any of thebeta-lactamases of the invention, including, for example, vectors,comprising such polynucleotides. Such polynucleotides may furthercomprise, in addition to sequences encoding the beta-lactamases of theinvention, one or more expression control elements. For example, thepolynucleotide, may comprise one or more promoters or transcriptionalenhancers, ribosomal binding sites, transcription termination signals,and polyadenylation signals, as expression control elements. Thepolynucleotide may be inserted within any suitable vector, which may becontained within any suitable host cell for expression. An illustrativepolyucleotide of the invention is SEQ ID NO: 7:

Full nucleotide sequence of A232G, A237S, A238G,S240D, and D276N mutant, Hind III site (AAGCTT-inbold) and additional K and T amino acids. Theleader and additional nucleotides (Hind III site and K and T amino acids - for the addition of theamino acid sequence QASKT) are underlined. SEQ ID NO: 7atgattcaaaaacgaaagcggacagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagcgcaagcttccaagacggagatgaaagatgattttgcaaaacttgaggaacaatttgatgcaaaactcgggatctttgcattggatacaggtacaaaccggacggtagcgtatcggccggatgagcgttttgcttttgcttcgacgattaaggctttaactgtaggcgtgcttttgcaacagaaatcaatagaagatctgaaccagagaataacatatacacgtgatgatcttgtaaactacaacccgattacggaaaagcacgttgatacgggaatgacgctcaaagagcttgcggatgcttcgcttcgatatagtgacaatgcggcacagaatctcattcttaaacaaattggcggacctgaaagtttgaaaaaggaactgaggaagattggtgatgaggttacaaatcccgaacgattcgaaccagagttaaatgaagtgaatccgggtgaaactcaggataccagtacagcaagagcacttgtcacaagccttcgagcctttgctcttgaagataaacttccaagtgaaaaacgcgagcttttaatcgattggatgaaacgaaataccactggagacgccttaatccgtgccggtgtgccggacggttgggaagtgggtgataaaactggaagcggagattatggaacccggaatgacattgccatcatttggccgccaaaaggagatcctgtcgttcttgcagtattatccagcagggataaaaaggacgccaagtatgataataaacttattgcagaggcaacaaaggtggtaatgaaagcctta aacatgaacggcaaataa

In some embodiments, the vector can remain episomal or becomechromosomally integrated, as long as the insert encoding the therapeuticagent can be transcribed. Vectors can be constructed by standardrecombinant DNA technology. Vectors can be, for example, plasmids,phages, cosmids, phagemids, viruses, or any other types known in theart, which are used for replication and expression in prokaryotic oreukaryotic cells (e.g. an adenovirus; a retrovirus; a lentivirus; anscAAV; pGEX vector; pET vector; and pHT vector). It will be appreciatedby one of skill in the art that a wide variety of components known inthe art (such as expression control elements) may be included in suchvectors, including a wide variety of transcription signals, such aspromoters and other sequences that regulate the binding of RNApolymerase onto the promoter. Any promoter known to be effective in thecells in which the vector will be expressed can be used to initiateexpression of the therapeutic agent. Suitable promoters may be inducibleor constitutive. Examples of suitable promoters include the pET system(INVITROGEN), lac promoter, tac, trc, T7, T7A3 promoter, PhoA, plux, andPhage lambda pR, lambda pL promoter (see, e.g. J Ind MicrobiolBiotechnol (2012) 39:383-399; Curr Opin Biotech 2001, 12: 195, thecontents of which are hereby incorporated by reference). The promotermay be inducible (e.g. via IPTG, metabolites, temperature). Examples ofsuitable promoters include the SV40 early promoter region, the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus, theHSV-1 thymidine kinase promoter, the regulatory sequences of themetallothionein gene, etc., as well as the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: elastase I gene control regionwhich is active in pancreatic acinar cells; insulin gene control regionwhich is active in pancreatic beta cells, immunoglobulin gene controlregion which is active in lymphoid cells, mouse mammary tumor viruscontrol region which is active in testicular, breast, lymphoid and mastcells, albumin gene control region which is active in liver,alpha-fetoprotein gene control region which is active in liver, alpha1-antitrypsin gene control region which is active in the liver,beta-globin gene control region which is active in erythroid cells,myelin basic protein gene control region which is active inoligodendrocyte cells in the brain, myosin light chain-2 gene controlregion which is active in skeletal muscle, and gonadotropin releasinghormone gene control region which is active in the hypothalamus.

The invention provides polynucleotides, such polynucleotides comprisinga nucleotide sequence encoding a beta-lactamase of the invention. Forexample, the nucleotide sequence that encodes a beta-lactamase of theinvention. Such polynucleotides may further comprise additional controlelement(s) operably linked to the nucleotide sequence, such as promoterelements and/or other transcription or expression-related signals. Thepolynucleotide may be inserted into various vectors, which may be usefulfor production of the therapeutic agent in host cells, including, forexample, bacterial and eukaryotic host cells. The beta-lactamase of theinvention can be prepared by known recombinant expression techniques.For example, to recombinantally produce the beta-lactamase of theinvention, a nucleic acid sequence encoding the respective gene isoperatively linked to a suitable promoter sequence such that the nucleicacid sequence encoding such beta-lactamase of the invention will betranscribed and/or translated in the host cells. Illustrative promotersare those useful for expression in various systems such as the T7promoter, Pspac, PgroES, Pgsi and Plux and amyQ promoter and/or amyQsignal peptide from Bacillus amyloliquefaciens (by way of non-limitingexample Gen Bank ID No. J01542.1, the contents of which are herebyincorporated by reference). Any commonly used expression system may beused, including eukaryotic or prokaryotic systems. Specific examplesinclude yeast (e.g., Saccharomyces spp., Pichia spp.), baculovirus,mammalian, and bacterial systems, such as E. coli, B. subtillis, andCaulobacter.

Host cells of the present invention include, for example, prokaryotic,eukaryotic, bacterial, yeast, algal, plant, insect, and/or mammaliancells. In some embodiments, the invention encompasses any type of cellthat recombinantly expresses genes associated with the invention,including prokaryotic and eukaryotic cells. In some embodiments the cellis a bacterial cell, such as, for example, Escherichia spp.,Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp.,Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacteriumspp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp.,Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp.,Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp.,Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillusspp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacterspp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp.,Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobiumspp., Saccharopolyspora spp., Agrobacterium spp. and Pantoea spp. Thebacterial cell can be a Gram-negative cell such as an Escherichia coli(E. coli) cell, or a Gram-positive cell such as a species of Bacillus.In other embodiments, the cell is a fungal cell such as, for example, ayeast cell, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichiaspp., Paffia spp., Kluyveromyces spp., Candida spp., Talaromyces spp.,Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp.,and industrial polyploid yeast strains. The yeast strain may be a S.cerevisiae strain or a Yarrowia spp. strain. Other examples of fungiinclude, for example, Aspergillus spp., Pennicilium spp., Fusarium spp.,Rhizopus spp., Acremonium spp., Neurospora spp., Sordaria spp.,Magnaporthe spp., Allomyces spp., Ustilago spp., Botrytis spp., andTrichoderma spp. In other embodiments, the cell is an algal cell or aplant cell (e.g., A. thaliana, C. reinhardtii, Arthrospira, P.tricomutum, T. suecica, P. carterae, P. tricomutum, Chlorella spp., suchas Chlorella vulgaris). Target cells can include transgenic andrecombinant cell lines. In addition, heterologous cell lines can beused, such as Chinese Hamster Ovary cells (CHO). Host cells may beunicellular host cells or multicellular host cells.

In various embodiments, the beta-lactamases of the invention possessfunctional characteristics that make them desirable for a variety ofuses, including therapeutic uses. Methods of characterizingbeta-lactamases are known in the art (e.g. nitrocefin assay as describedby O'Callaghan, et al. Antimicrob. Agents Chemother, 1:283-288; thevarious methods of Viswanatha et al. Methods Mol Med. 2008; 142:239-60).

In one embodiment, the inventive beta-lactamases hydrolyze one or moreof penicillins and cephalosporins. As used throughout, penicillinsinclude, for example, Amoxicillin (e.g. NOVAMOX, AMOXIL); Ampicillin(e.g. PRINCIPEN); Azlocillin; Carbenicillin (e.g. GEOCILLIN);Cloxacillin (e.g. TEGOPEN); Dicloxacillin (e.g. DYNAPEN); Flucloxacillin(e.g. FLOXAPEN); Mezlocillin (e.g. MEZLIN); Methicillin (e.g.STAPHCILLIN); Nafcillin (e.g. UNIPEN); Oxacillin (e.g. PROSTAPHLIN);Penicillin G (e.g. PENTIDS or PFIZERPEN); Penicillin V (e.g. VEETIDS(PEN-VEE-K)); Piperacillin (e.g. PIPRACIL); Temocillin (e.g. NEGABAN);and Ticarcillin (e.g. TICAR). As used throughout, cephalosporinsinclude, for example, a first generation cephalosporin (e.g. Cefadroxil(e.g. DURICEF); Cefazolin (e.g. ANCEF); Ceftolozane,Cefalotin/Cefalothin (e.g. KEFLIN); Cefalexin (e.g. KEFLEX); a secondgeneration cephalosporin (e.g. Cefaclor (e.g. DISTACLOR); Cefamandole(e.g. MANDOL); Cefoxitin (e.g. MEFOXIN); Cefprozil (e.g. CEFZIL);Cefuroxime (e.g. CEFTIN, ZINNAT)); a third generation cephalosporin(e.g. Cefixime (e.g. SUPRAX); Cefdinir (e.g. OMNICEF, CEFDIEL);Cefditoren (e.g. SPECTRACEF); Cefoperazone (e.g. CEFOBID); Cefotaxime(e.g. CLAFORAN); Cefpodoxime (e.g. VANTIN); Ceftazidime (e.g. FORTAZ);Ceftibuten (e.g. CEDAX) Ceftizoxime (e.g. CEFIZOX); and Ceftriaxone(e.g. ROCEPHIN)); a fourth generation cephalosporin (e.g. Cefepime (e.g.MAXIPIME)); or a fifth generation cephalosporin (e.g. Ceftarolinefosamil (e.g. TEFLARO); Ceftobiprole (e.g. ZEFTERA)). In a specificembodiment, cephalosporins include, for example, cefoperazone,ceftriaxone or cefazolin. In a specific embodiment, the inventivebeta-lactamases have improved catalytic efficiency againstcephalosporins as compared to SEQ ID NO: 1.

In various embodiments, the inventive beta-lactamases possess desirableenzyme kinetic characteristics. For example, in some embodiments, thebeta-lactamases possess a low K_(M) for at least one cephalosporin,including, for example, a K_(M) of less than about 500 μM, or about 100μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100 nM), or about0.01 μM (10 nM), or about 1 nM. For example, in some embodiments, thebeta-lactamases possess a low K_(M) for at least one penicillin,including, for example, a K_(M) of less than about 500 μM, or about 100μM, or about 10 μM, or about 1 μM, or about 0.1 μM (100 nM), or about0.01 μM (10 nM), or about 1 nM. In various embodiments, the inventivebeta-lactamases possess a high V_(max) for at least one cephalosporin,including, for example, V_(max) which is greater than about 100 s-1, orabout 1000 s-1, or about 10000 s-1, or about 100000 s-1, or about1000000 s-1. In various embodiments, the inventive beta-lactamasespossess a high V_(max) for at least one penicillin, including, forexample, V_(max) which is greater than about 100 s-1, or about 1000 s-1,or about 10000 s-1, or about 100000 s-1, or about 1000000 s-1. Invarious embodiments, the inventive beta-lactamases possess catalyticefficiency is greater than about 10⁶ M⁻¹ s⁻¹ for at least onecephalosporin. In various embodiments, the inventive beta-lactamasespossess catalytic efficiency is greater than about 10⁶ M⁻¹ s⁻¹ for atleast one penicillin. In various embodiments, the inventivebeta-lactamases possess the desirable enzyme kinetic characteristics forat least one of either or both of cephalosporins and penicillins.

In various embodiments, the inventive beta-lactamases are stable and/oractive in the GI tract, e.g. in one or more of the mouth, esophagus,stomach, duodenum, small intestine, duodenum, jejunum, ileum, largeintestine, colon transversum, colon descendens, colon ascendens, colonsigmoidenum, cecum, and rectum. In a specific embodiment, thebeta-lactamase is stable in the large intestine, optionally selectedfrom one or more of colon transversum, colon descendens, colonascendens, colon sigmoidenum and cecum. In a specific embodiment, thebeta-lactamase is stable in the small intestine, optionally selectedfrom one or more of duodenum, jejunum, and ileum. In some embodiments,the beta-lactamase is resistant to proteases in the GI tract, includingfor example, the small intestine. In some embodiments, thebeta-lactamase is substantially active at a pH of about 6.0 to about7.5, e.g. about 6.0, or about 6.1, or about 6.2, or about 6.3, or about6.4, or about 6.5, or about 6.6, or about 6.7, or about 6.8, or about6.9, or about 7.0, or about 7.1, or about 7.2, or about 7.3, or about7.4, or about 7.5 (including, for example, via formulation, as describedherein). In various embodiments, the beta-lactamases of the presentinvention are resistant to one or more beta-lactamase inhibitors,optionally selected from avibactam, tazobactam, sulbactam, andclavulanic acid. In some embodiments, stable refers to an enzyme thathas a long enough half life and maintains enough activity fortherapeutic effectiveness.

In various embodiments, the present invention pertains to pharmaceuticalcompositions comprising a beta-lactamase described herein and one ormore of and a pharmaceutically acceptable carrier or excipient asdescribed herein. In a specific embodiment, the pharmaceuticalcomposition is formulated for oral administration, e.g. as a tablet ormulti-particulate sprinkle or a multi-particulate capsule. However, asdescribed herein, other administration routes and formulations are alsoprovided.

In some embodiments, the pharmaceutical compositions may be used inconjunction with or be co-formulations with an additional agent. In someembodiments, the additional agent is an additional antibioticdegradation enzyme, such as, for example, a beta-lactamase of class EC3.5.2.6. In some embodiments, the antibiotic degradation enzyme isselected from a functional Group 1, Group 2, Group 3, or a Group 4beta-lactamase (see, e.g., Bush et al., Antimicrob. Agents Chemother,39: 1211, the contents of which are hereby incorporated by reference):without wishing to be bound by theory, Group 1 consists ofcephalosporinases that are not well inhibited by clavulanic acid; Group2 consists of penicillinases, cephalosporinases and broad-spectrumbeta-lactamases that are generally inhibited by active site-directedbeta-lactamase inhibitors; Group 3 consists of metallo-beta-lactamasesthat hydrolyze penicillins, cephalosporins and carbapenems, and that arepoorly inhibited by almost all beta-lactam-containing molecules; andGroup 4 consists of penicillinases that are not well inhibited byclavulanic acid) and/or a molecular/Ambler class A, or class B, or classC, or class D beta-lactamase (see, e.g., Ambler 1980, Philos Trans R SocLond B Biol Sci. 289: 321 the contents of which are hereby incorporatedby reference), without wishing to be bound by theory: Classes A, C, andD gather evolutionarily distinct groups of serine beta-lactamaseenzymes, and class B the zinc-dependent (“EDTA-inhibited”)beta-lactamase enzymes (see Ambler R. P. et al., 1991, Biochem J. 276:269-270, the contents of which are hereby incorporated by reference). Insome embodiments, the antibiotic degradation enzyme is a serinebeta-lactamase or a zinc-dependent (EDTA-inhibited) beta-lactamase. Forexample, in some embodiments, the beta-lactamase is one or more of P1A,P2A, or P3A. Further, the beta-lactamase may be an extended-spectrumbeta-lactamase (ESBL), optionally selected from a TEM, SHV, CTX-M, OXA,PER, VEB, GES, and IBC beta-lactamase. Further, the beta-lactamase maybe an inhibitor-resistant β-lactamase, optionally selected from anAmpC-type β-lactamases, Carbapenemase, IMP-type carbapenemases(metallo-β-lactamases), VIM (Verona integron-encodedmetallo-β-lactamase), OXA (oxacillinase) group of β-lactamases, KPC (K.pneumonia carbapenemase), CMY (Class C), SME, IMI, NMC and CcrA, and aNDM (New Delhi metallo-β-lactamase, e.g. NDM-1) beta-lactamase.

In some embodiments, the additional agent is an adjunctive therapy thatis used in, for example, the treatment of CDI as described herein. Insome embodiments, the additional agent is metronidazole (e.g. FLAGYL),fidaxomicin (e.g. DIFICID), or vancomycin (e.g. Vancocin), rifaximin,fecal bacteriotherapy, charcoal-based binders (e.g. DAV132), probiotictherapy (see, e.g., Intnat'l J Inf Dis, 16 (11): e786, the contents ofwhich are hereby incorporated by reference, illustrative probioticsinclude Saccharomyces boulardii; Lactobacillus rhamnosus GG;Lactobacillus plantarum 299v; Clostridium butyricum M588; Clostridiumdifficile VP20621 (non-toxigenic C. difficile strain); combination ofLactobacillus casei, Lactobacillus acidophilus (Bio-K+CL1285);combination of Lactobacillus casei, Lactobacillus bulgaricus,Streptococcus thermophilus (Actimel); combination of Lactobacillusacidophilus, Bifidobacterium bifidum (Florajen3); combination ofLactobacillus acidophilus, Lactobacillus bulgaricus delbrueckii subsp.bulgaricus, Lactobacillus bulgaricus casei, Lactobacillus bulgaricusplantarum, Bifidobacterium longum, Bifidobacterium infantis,Bifidobacterium breve, Streptococcus salivarius subsp. thermophilus(VSL#3)) and antibody or other biologic therapy (e.g. monoclonalantibodies against C. difficile toxins A and B as described in N Engl JMed. 2010; 362(3):197, the content of which are hereby incorporated byreference in their entirety; neutralizing binding proteins, for example,arranged as multimers, which are directed to one or more of SEQ ID NOs.recited in United States Patent Publication No. 2013/0058962 (e.g. oneor more of SEQ ID Nos.: 59, 60, 95, 67, 68, and 87), the contents ofwhich are hereby incorporated by reference); or any neutralizing bindingprotein directed against C. difficile binary toxin. In some embodiments,any of the penicillins and cephalosporins described herein may be theadditional agent.

For all additional agent compositions and methods, targeting to variousparts of the GI tract may be employed as described herein.

In some embodiments, the inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein, includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the composition such that covalent attachment doesnot prevent the activity of the composition. For example, but not by wayof limitation, derivatives include composition that have been modifiedby, inter alia, glycosylation, lipidation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of turicamycin,etc. Additionally, the derivative can contain one or more non-classicalamino acids.

In still other embodiments, the inventive beta-lactamases and/orpharmaceutical compositions described herein may be modified to addeffector moieties such as chemical linkers, detectable moieties such asfor example fluorescent dyes, enzymes, substrates, bioluminescentmaterials, radioactive materials, and chemiluminescent moieties, orfunctional moieties such as for example streptavidin, avidin, biotin, acytotoxin, a cytotoxic agent, and radioactive materials.

The inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) described herein can possess a sufficiently basicfunctional group, which can react with an inorganic or organic acid, ora carboxyl group, which can react with an inorganic or organic base, toform a pharmaceutically acceptable salt. A pharmaceutically acceptableacid addition salt is formed from a pharmaceutically acceptable acid, asis well known in the art. Such salts include the pharmaceuticallyacceptable salts listed in, for example, Journal of PharmaceuticalScience, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts;Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.),Verlag, Zurich (Switzerland) 2002, which are hereby incorporated byreference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thecompositions of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-0H-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Further, any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein can beadministered to a subject as a component of a composition that comprisesa pharmaceutically acceptable carrier or vehicle. Such compositions canoptionally comprise a suitable amount of a pharmaceutically acceptableexcipient so as to provide the form for proper administration.

Pharmaceutical excipients can be liquids, such as water and oils,including those of petroleum, animal, vegetable, or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The pharmaceutical excipients can be, for example, saline, gum acacia,gelatin, starch paste, talc, keratin, colloidal silica, urea and thelike. In addition, auxiliary, stabilizing, thickening, lubricating, andcoloring agents can be used. In one embodiment, the pharmaceuticallyacceptable excipients are sterile when administered to a subject. Wateris a useful excipient when any agent described herein is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid excipients, specifically forinjectable solutions. Suitable pharmaceutical excipients also includestarch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene, glycol, water, ethanoland the like. Any agent described herein, if desired, can also compriseminor amounts of wetting or emulsifying agents, or pH buffering agents.

The present invention includes the described inventive beta-lactamasesand/or pharmaceutical compositions (and/or additional agents) in variousformulations. Any inventive beta-lactamase and/or pharmaceuticalcomposition (and/or additional agents) described herein can take theform of solutions, suspensions, emulsion, drops, tablets, pills,pellets, capsules, capsules containing liquids, powders,sustained-release formulations, suppositories, emulsions, aerosols,sprays, suspensions, or any other form suitable for use. In oneembodiment, the composition is in the form of a capsule (see, e.g., U.S.Pat. No. 5,698,155). Other examples of suitable pharmaceuticalexcipients are described in Remington's Pharmaceutical Sciences1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995), incorporated hereinby reference.

Where necessary, the inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) can also include a solubilizingagent. Also, the agents can be delivered with a suitable vehicle ordelivery device as known in the art. Compositions for administration canoptionally include a local anesthetic such as, for example, lignocaineto lessen pain at the site of the injection. Combination therapiesoutlined herein can be co-delivered in a single delivery vehicle ordelivery device.

The formulations comprising the inventive beta-lactamases and/orpharmaceutical compositions (and/or additional agents) of the presentinvention may conveniently be presented in unit dosage forms and may beprepared by any of the methods well known in the art of pharmacy. Suchmethods generally include the step of bringing the therapeutic agentsinto association with a carrier, which constitutes one or more accessoryingredients. Typically, the formulations are prepared by uniformly andintimately bringing the therapeutic agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into dosage forms of the desiredformulation (e.g., wet or dry granulation, powder blends, etc., followedby tableting using conventional methods known in the art)

In one embodiment, any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein is formulatedin accordance with routine procedures as a composition adapted for amode of administration described herein.

In some embodiments, the administration of any inventive beta-lactamasesand/or pharmaceutical compositions (and/or additional agents) is any oneof oral, intravenous, and parenteral. In some embodiments, theadministration of any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) is not intravenous in order to,for example, prevent interference with an antibiotic administeredsystemically. In other embodiments, routes of administration include,for example: oral, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal,intracerebral, intravaginal, transdermal, rectally, by inhalation, ortopically, particularly to the ears, nose, eyes, or skin. In someembodiments, the administering is effected orally or by parenteralinjection. The mode of administration can be left to the discretion ofthe practitioner, and depends in-part upon the site of the medicalcondition. In most instances, administration results in the release ofany agent described herein into the bloodstream.

Any inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) described herein can be administered orally. Suchinventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) can also be administered by any other convenientroute, for example, by intravenous infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and can be administeredtogether with an additional agent. Administration can be systemic orlocal. In some embodiments, administration is not at the site ofinfection to avoid, for example, hydrolysis of an antibiotic at the siteof infection. Various delivery systems are known, e.g., encapsulation inliposomes, microparticles, microcapsules, capsules, etc., and can beused to administer.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment.

In one embodiment, any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein is formulatedin accordance with routine procedures as a composition adapted for oraladministration to humans. Compositions for oral delivery can be in theform of tablets, lozenges, aqueous or oily suspensions, granules,powders, sprinkles, emulsions, capsules, syrups, or elixirs, forexample. Orally administered compositions can comprise one or moreagents, for example, sweetening agents such as fructose, aspartame orsaccharin; flavoring agents such as peppermint, oil of wintergreen, orcherry; coloring agents; and preserving agents, to provide apharmaceutically palatable preparation. Moreover, where in tablet orpill form, the compositions can be coated to delay disintegration toprovide a sustained action over an extended period of time. Selectivelypermeable membranes surrounding an osmotically active agent driving anyinventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) described herein are also suitable for orallyadministered compositions. In these latter platforms, fluid from theenvironment surrounding the capsule is imbibed by the driving compound,which swells to displace the agent or agent composition through anaperture. These delivery platforms can provide an essentially zero orderdelivery profile as opposed to the spiked profiles of immediate releaseformulations. A time-delay material such as glycerol monostearate orglycerol stearate can also be useful. Oral compositions can includestandard excipients such as mannitol, lactose, starch, magnesiumstearate, sodium saccharin, cellulose, ethacrylic acid and derivativepolymers thereof, and magnesium carbonate. In one embodiment, theexcipients are of pharmaceutical grade. Suspensions, in addition to theactive compounds, may contain suspending agents such as, for example,ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitanesters, microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, tragacanth, etc., and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art.

Inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) may be administered to a subject, by, for example,directly or indirectly contacting the mucosal tissues of thegastrointestinal tract. The gastrointestinal tract a includes organs ofthe digestive system such as mouth, esophagus, stomach, duodenum, smallintestine, large intestine and rectum and includes all subsectionsthereof (e.g. the small intestine may include the duodenum, jejunum andileum; the large intestine may include the colon transversum, colondescendens, colon ascendens, colon sigmoidenum and cecum). Variousmethods may be used to formulate and/or deliver the agents descriedherein to a location of interest. For example, the inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) described herein may be formulated for delivery to one or moreof the stomach, small intestine, large intestine and rectum and includesall subsections thereof (e.g. duodenum, jejunum and ileum, colontransversum, colon descendens, colon ascendens, colon sigmoidenum andcecum). In some embodiments, the compositions described herein may beformulated to deliver to the upper or lower GI tract.

In some embodiments, the compositions of the invention are formulatedfor enteric delivery. For example, the compositions may be formulated ascapsules or tablets for oral delivery, and may comprise adelayed-release coating containing one or more enteric agents. Adelayed-release coating is substantially stable in gastric fluid andsubstantially unstable (e.g., dissolves rapidly or is physicallyunstable) in intestinal fluid, thus providing for substantial release ofthe active agent from the composition in the affected region of thesmall intestine, e.g., the duodenum, the jejunum, and/or the ileum orlarge intestine, e.g. the colon transversum, colon descendens, colonascendens, colon sigmoidenum and cecum.

The inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) are generally stable in gastric fluid or simulatedintestinal fluid, that is, the compositions are stable in acidicenvironments. Thus, the compositions release less than 30% by weight ofthe active agent in gastric fluid with a pH of 5 or less, or simulatedgastric fluid with a pH of 5 or less, in approximately 120 minutes.Compositions of the invention may release from about 0% to about 25%, orfrom about 0% to about 10% by weight of the active agent in gastricfluid with a pH of 5 or less, or simulated gastric fluid with a pH of 5or less, in approximately 120 minutes. Compositions of the invention incertain embodiments release no more than about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%by weight of the active agent in gastric fluid, or simulated gastricfluid with a pH of 5 or less, in approximately 120 minutes. Thepharmaceutical compositions generally release active agent to actlocally at regions of the small or large intestine. In certainembodiments, the composition of the invention release about 70% or moreby weight of the active agent in the small or large intestine withinabout 120 minutes. In certain embodiments, the composition releases 80%or more, or 90% or more, of the active agent in small or largeintestine, within about ninety minutes or within about 120 minutes. Incertain embodiments this release in the small or large intestine ismediated by pH of gastric fluid or simulated gastric fluid—for example,a release when the pH of about 5 or greater (e.g. pH of about 5.5-6.5for release in the duodenum, pH of about 6-7 for release in the colonascendens or jejunum, pH of about 6.5-7 for release in the ileum, pH ofabout 7-7.5 for release in the colon descendens).

The pharmaceutical composition may control intestinal release of theactive agent through one or more delayed-release coating(s), whichremain essentially intact, and/or which may be essentially insoluble, ingastric fluid. The stability of the delayed-release coating can be pHdependent. Delayed-release coatings that are pH dependent will besubstantially stable in acidic environments (pH 5 or less), andsubstantially unstable in near neutral to alkaline environments (pHgreater than 5). For example, the delayed-release coating mayessentially disintegrate or dissolve in near neutral to alkalineenvironments, such as are found in the small intestine or largeintestine, to thereby release active agent locally to diseased oraffected tissue.

Examples of simulated gastric fluid and simulated intestinal fluidinclude, but are not limited to, those disclosed in the 2005Pharmacopeia 23NF/28USP in Test Solutions at page 2858 and/or othersimulated gastric fluids and simulated intestinal fluids known to thoseof skill in the art, for example, simulated gastric fluid and/orintestinal fluid prepared without enzymes.

Alternatively, the stability of the delayed-release coating may beenzyme-dependent. Delayed-release coatings that are enzyme dependentwill be substantially stable in fluid that does not contain a particularenzyme and substantially unstable in fluid containing the enzyme. Thedelayed-release coating will essentially disintegrate or dissolve influid containing the appropriate enzyme. Enzyme-dependent control can bebrought about, for example, by using materials which release the activeingredient only on exposure to enzymes in the intestine, such asgalactomannans.

In some embodiments, the target organ for the release of the activeagent from the compositions of the invention, which include gastricresistant capsules or tablets, is the small intestine, such as theduodenum and the jejunum or the large intestine, e.g. the colontransversum, colon descendens, colon ascendens, colon sigmoidenum andcecum. See Remington's Pharmaceutical Sciences, 16th Ed., Eds. Osol,Mack Publishing Co., Chapter 89 (1980); Digenis et al., J. Pharm. Sci.,83:915-921 (1994); Vantini et al., Clinica Terapeutica, 145:445-451(1993); Yoshitomi et al., Chem. Pharm. Bull., 40:1902-1905 (1992); Thomaet al., Pharmazie, 46:331-336 (1991); Morishita et al., Drug Design andDelivery, 7:309-319 (1991); and Lin et al., Pharmaceutical Res.,8:919-924 (1991) for examples of the preparation of such tablets orcapsules (the contents of the above are hereby incorporated by referencein their entireties).

In some embodiments, the compositions of the present invention may beformulated using the EUDRAGIT system, as known in the art and describedin Pharma Polymer No. 7, October 2000, the contents of which are herebyincorporated by reference in their entirety.

The dosage of any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein as well as thedosing schedule can depend on various parameters, including, but notlimited to, the disease being treated, the subject's general health, andthe administering physician's discretion. Any agent described herein,can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksafter) the administration of an additional therapeutic agent, to asubject in need thereof. In various embodiments any agent describedherein is administered 1 minute apart, 10 minutes apart, 30 minutesapart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hoursapart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24hours apart or no more than 48 hours apart.

The amount of any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein that is admixedwith the carrier materials to produce a single dosage can vary dependingupon the subject being treated and the particular mode ofadministration. In vitro or in vivo assays can be employed to helpidentify optimal dosage ranges.

In general, the doses that are useful are known to those in the art. Forexample, doses may be determined with reference Physicians' DeskReference, 66th Edition, PDR Network; 2012 Edition (Dec. 27, 2011), thecontents of which are incorporated by reference in its entirety. In someembodiment, the present invention allows a patient to receive doses thatexceed those determined with reference Physicians' Desk Reference.

The dosage of any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein can depend onseveral factors including the severity of the condition, whether thecondition is to be treated or prevented, and the age, weight, and healthof the subject to be treated. Additionally, pharmacogenomic (the effectof genotype on the pharmacokinetic, pharmacodynamic or efficacy profileof a therapeutic) information about a particular subject may affectdosage used. Furthermore, the exact individual dosages can be adjustedsomewhat depending on a variety of factors, including the specificcombination of the agents being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the particular disease beingtreated, the severity of the disorder, and the anatomical location ofthe disorder. Some variations in the dosage can be expected.

In specific embodiments, the concentration of any inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) is about 0.2 to about 20 g/L when administered orally or about 4g/mL when administered intravenously.

In some embodiments, when orally administered to a mammal, the dosage ofany inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) described herein may be about 0.001 mg/kg/day toabout 100 mg/kg/day, about 0.01 mg/kg/day to about 50 mg/kg/day, orabout 0.1 mg/kg/day to about 10 mg/kg/day. When orally administered to ahuman, the dosage of any agent described herein is normally about 0.001mg to 1000 mg per day, about 1 mg to about 600 mg per day, or about 5 mgto about 30 mg per day. In one embodiment, 100 mg per day of theinventive beta-lactamases and/or pharmaceutical compositions isadministered orally to a human. For administration of any inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) described herein by parenteral injection, the dosage is normallyabout 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day,or about 3 mg to about 5 mg per day. Injections may be given up to fourtimes daily. Generally, when orally or parenterally administered, thedosage of any agent described herein is normally about 0.1 mg to about1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mgto about 5 mg per day. A dosage of up to about 3000 mg per day can beadministered. In some embodiments, the dose may be about 1000 mg perday. In some embodiments, the following dose regiment can be used: 100mg, four times a day.

In various embodiments, the dosage of any inventive beta-lactamasesand/or pharmaceutical compositions (and/or additional agents) describedherein is administered to achieve an intestinal concentration of fromabout 1 ng/ml to about 10,000 ng/ml, about 1 ng/ml to about 5,000 ng/ml,about 1 ng/ml to about 2,500 ng/ml, about 1 ng/ml to about 1,000 ng/ml,about 1 ng/ml to about 500 ng/ml, about 1 ng/ml to about 250 ng/ml,about 1 ng/ml to about 100 ng/ml, about 1 ng/ml to about 50 ng/ml, about1 ng/ml to about 25 ng/ml, about 1 ng/ml to about 10 ng/ml, about 10ng/ml to about 10,000 ng/ml, about 10 ng/ml to about 5,000 ng/ml, about10 ng/ml to about 2,500 ng/ml, about 10 ng/ml to about 1,000 ng/ml,about 10 ng/ml to about 500 ng/ml, about 10 ng/ml to about 250 ng/ml,about 10 ng/ml to about 100 ng/ml, about 10 ng/ml to about 50 ng/ml,about 10 ng/ml to about 25 ng/ml, about 100 ng/ml to about 10,000 ng/ml,about 100 ng/ml to about 5,000 ng/ml, about 100 ng/ml to about 2,500ng/ml, about 100 ng/ml to about 1,000 ng/ml, about 100 ng/ml to about500 ng/ml, or about 100 ng/ml to about 250 ng/ml. In variousembodiments, the intestinal concentration of any inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) described herein is 10 ng/ml, 100 ng/ml, or 1,000 ng/ml.

In another embodiment, delivery can be in a vesicle, in particular aliposome (see Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989).

Any inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) described herein can be administered bycontrolled-release or sustained-release means or by delivery devicesthat are well known to those of ordinary skill in the art. Examplesinclude, but are not limited to, those described in U.S. Pat. Nos.3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533;5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and5,733,556, each of which is incorporated herein by reference in itsentirety. Such dosage forms can be useful for providing controlled- orsustained-release of one or more active ingredients using, for example,hydropropyl cellulose, hydropropylmethyl cellulose,polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes,osmotic systems, multilayer coatings, microparticles, liposomes,microspheres, or a combination thereof to provide the desired releaseprofile in varying proportions. Suitable controlled- orsustained-release formulations known to those skilled in the art,including those described herein, can be readily selected for use withthe active ingredients of the agents described herein. The inventionthus provides single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled- or sustained-release.

Controlled- or sustained-release of an active ingredient can bestimulated by various conditions, including but not limited to, changesin pH, changes in temperature, stimulation by an appropriate wavelengthof light, concentration or availability of enzymes, concentration oravailability of water, or other physiological conditions or compounds.

In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al, 1989, J. Neurosurg. 71:105).

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Administration of any inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) described herein can,independently, be one to six times daily (e.g. 1, or 2, or 3, or 4, or5, or 6 times per day) or one to four times per month or one to sixtimes per year or once every two, three, four or five years.Administration can be for the duration of one day or one month, twomonths, three months, six months, one year, two years, three years, andmay even be for the life of the subject. Chronic, long-termadministration will be indicated in many cases. The dosage may beadministered as a single dose or divided into multiple doses. Ingeneral, the desired dosage should be administered at set intervals fora prolonged period, usually at least over several weeks or months,although longer periods of administration of several months or years ormore may be needed.

The dosage regimen utilizing any inventive beta-lactamases and/orpharmaceutical compositions (and/or additional agents) described hereincan be selected in accordance with a variety of factors including type,species, age, weight, sex and medical condition of the subject; theseverity of the condition to be treated; the route of administration;the renal or hepatic function of the subject; the pharmacogenomic makeupof the individual; and the specific compound of the invention employed.Any inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) described herein can be administered in a singledaily dose, or the total daily dosage can be administered in divideddoses of two, three or four times daily. Furthermore, any inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) described herein can be administered continuously rather thanintermittently throughout the dosage regimen.

In various aspects, the present invention provides methods for treatingor preventing an antibiotic-induced adverse effect in the GI tract,comprising administering an effective amount of a beta-lactamase and/orpharmaceutical composition (and/or additional agent) described herein toa patient in need thereof. In one aspect, the present invention providesmethods for preventing an antibiotic-induced adverse effect in the GItract, comprising administering an effective amount of a beta-lactamaseand/or pharmaceutical composition (and/or additional agent) describedherein to a patient in need thereof (by way of non-limiting example, apatient that is being administered or will be administered anantibiotic, including those described herein). In various aspects, thepresent invention provides methods for treating or preventing C.difficile infection (CDI) and/or a C. difficile-associated disease,comprising administering an effective amount of a beta-lactamase and/orpharmaceutical composition (and/or additional agent) described herein toa patient in need thereof. In one aspect, the present invention providesmethods for preventing C. difficile infection (CDI) and/or a C.difficile-associated disease, comprising administering an effectiveamount of a beta-lactamase and/or pharmaceutical composition (and/oradditional agent) described herein to a patient in need thereof (by wayof non-limiting example, a patient that is being administered or will beadministered an antibiotic, including those described herein.

In various embodiments, the antibiotic-induced adverse effect and/or CDIor C. difficile-associated disease is one or more of:antibiotic-associated diarrhea, C. difficile diarrhea (CDD), C.difficile intestinal inflammatory disease, colitis, pseudomembranouscolitis, fever, abdominal pain, dehydration and disturbances inelectrolytes, megacolon, peritonitis, and perforation and/or rupture ofthe colon.

In various aspects, the present invention provides methods forprotecting a subject's gastrointestinal microbiome, comprisingadministering an effective amount of a beta-lactamase and/orpharmaceutical composition (and/or additional agent) described herein.In various embodiments, the subject is undergoing treatment or hasrecently undergone treatment with an antibiotic. In variousembodiements, the beta-lactamase and/or pharmaceutical composition(and/or additional agent) described herein is capable of degrading orinactivating the antibiotic.

In various embodiments, the subjects include, but are not limited to,subjects that are at a particular risk for a microbiome-mediateddisorder, such as, by way of non-limiting example, those undergoingtreatment or having recently undergone treatment with an antibiotic. Forexample, the subject may have taken an antibiotic during the past about30 or so days and/or have an immune system that is weak (e.g. from achronic illness) and/or is a women and/or is elderly (e.g. over about 65years old) and/or is an elderly woman and/or is undergoing (or hasundergone) treatment with for heartburn or stomach acid disorders (e.g.with agents such as PREVACID, TAGAMET, PRILOSEC, or NEXIUM and relateddrugs) and/or has recently been in the hospital, including in anintensive care unit, or lives in a nursing home. Accordingly, in someembodiments, the methods and uses of the present invention treat orprevent a nosocomial infection and/or a secondary emergent infectionand/or a hospital acquired infection (HAI).

In various embodiments, the methods of the invention comprise treatingor preventing a microbiome-mediated disorder. Illustrativemicrobiome-mediated disorder includes, but are not limited to, forexample, those found in Table 3 of WO 2014/121298, the entire contentsof which are incorporated herein by reference. For example, themicrobiome-mediated disorder may be selected from an antibiotic-inducedadverse effect, a C. difficile infection (CDI), a C.difficile-associated disease, ulcerative colitis, Crohn's disease, andirritable bowel syndrome. In various embodiments, themicrobiome-mediated disorder is an antibiotic-induced adverse effect, aC. difficile infection (CDI), or a C. difficile-associated disease. Inan embodiment, the present invention provides methods for treating anantibiotic-induced adverse effect in the GI tract, comprisingadministering an effective amount of a beta-lactamase and/orpharmaceutical composition (and/or additional agent) described herein toa subject who is undergoing treatment or has recently undergonetreatment with an antibiotic. In another embodiment, the presentinvention provides methods for preventing an antibiotic-induced adverseeffect in the GI tract, comprising administering an effective amount ofa beta-lactamase and/or pharmaceutical composition (and/or additionalagent) described herein to a subject who is undergoing treatment or hasrecently undergone treatment with an antibiotic.

In various embodiments, the present uses and methods pertain toco-treatment (simultaneously or sequentially) with the beta-lactamasesand/or pharmaceutical compositions of the present invention and anyadditional agent described herein and/or any initial and/or adjunctivetherapy, or treatment with a co-formulation of the beta-lactamasesand/or pharmaceutical compositions of the present invention and anyadditional agent described herein and/or any initial and/or adjunctivetherapy for treatment of the various diseases described herein, ormethods of treating the various diseases described herein in a patientundergoing treatment with any additional agent described herein and/orany initial and/or adjunctive therapy described herein by administeringa beta-lactamases and/or pharmaceutical compositions of the presentinvention to the patient.

In various embodiments, the CDI and/or C. difficile associated diseaseis treated or prevented in the context of initial onset orrelapse/recurrence (e.g. due to continued or restarted antbiotictherapy). For example, in a patient that has previously suffered fromCDI, the present beta-lactamases and/or pharmaceutical compositions(and/or additional agents) may be administered upon the first symptomsof recurrence. By way of non-limiting example, symptoms of recurrenceinclude, in a mild case, about 5 to about 10 watery bowel movements perday, no significant fever, and only mild abdominal cramps while bloodtests may show a mild rise in the white blood cell count up to about15,000 (normal levels are up to about 10,000), and, in a severe case,more than about 10 watery stools per day, nausea, vomiting, high fever(e.g. about 102-104° F.), rectal bleeding, severe abdominal pain (e.g.with tenderness), abdominal distention, and a high white blood count(e.g. of about 15,000 to about 40,000).

Regardless of initial onset or relapse/recurrence, CDI and/or C.difficile associated disease may be diagnosed via any of the symptomsdescribed herein (e.g. watery diarrhea about 3 or more times a day forabout 2 days or more, mild to bad cramping and pain in the belly, fever,blood or pus in the stool, nausea, dehydration, loss of appetite, lossof weight, etc.). Regardless of initial onset or relapse/recurrence, CDIand/or C. difficile associated disease may also be diagnosed via enzymeimmunoassays e.g. to detect the C. difficile toxin A or B antigen and/orglutamine dehydrogenase (GDH), which is produced by C. difficileorganisms), polymerase chain reaction (e.g. to detect the C. difficiletoxin A or B gene or a portion thereof (e.g. tcdA or tcdB), includingthe ILLUMIGENE LAMP assay), a cell cytotoxicity assay. For example, anyone of the following tests may be used may be used: Meridian ImmunoCardToxins A/B; Wampole Toxin A/B Quik Chek; Wampole C. diff Quik ChekComplete; Remel Xpect Clostridium difficile Toxin A/B; Meridian PremierToxins A/B; Wampole C. difficile Tox A/B II; Remel Prospect Toxin A/BEIA; Biomerieux Vidas C. difficile Toxin A&B; BD Geneohm C. diff;Prodesse Progastro CD; and Cepheid Xpert C. diff. In variousembodiments, the clinical sample is a patient stool sample.

Also a flexible sigmoidoscopy “scope” test and/or an abdominal X-rayand/or a computerized tomography (CT) scan, which provides images ofyour colon, may be used in assessing a patient (e.g. looking forcharacteristic creamy white or yellow plaques adherent to the wall ofthe colon). Further, biopsies (e.g. of any region of the GI tract) maybe used to assess a potential CDI and/or C. difficile associated diseasepatient.

Furthermore, the patients of the invention include, but are not limitedto, patients that are at a particular risk for CDI and/or C. difficileassociated disease, such as those which have been taking an antibioticduring the past 30 or so days and/or have an immune system that is weak(e.g. from a chronic illness) and/or are women and/or are elderly (e.g.over about 65 years old) and/or are elderly woman and/or undergotreatment with for heartburn or stomach acid disorders (e.g. with agentssuch as PREVACID, TAGAMET, PRILOSEC, or NEXIUM and related drugs) and/orhave recently been in the hospital, including in an intensive care unit,or live in a nursing home. Accordingly, in some embodiments, the methodsand uses of the present invention treat or prevent a nosocomialinfection and/or a secondary emergent infection and/or a hospitalacquired infection (HAI).

In some embodiments, the methods of the invention treat or prevent aceftriaxone-associated adverse effect (e.g. diarrhea, nausea, vomiting,dysgeusia, and pseudomembranous colitis disease and/or symptoms).

In some embodiments, the methods and uses of the present inventionrelate to a patient is undergoing treatment or has recently undergonetreatment with one or more primary antibiotic. A “primary antibiotic”refers to an antibiotic that is administered to a patient and which mayresult in CDI and/or C. difficile associated disease. These include theantibiotics that most often lead to CDI and/or C. difficile associateddisease: fluoroquinolones, cephalosporins, clindamycin and penicillins.

In some embodiments, the methods and uses of the present inventionrelate to the inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) which hydrolyze a primaryantibiotic before it enters the large intestine. In some embodiments,the methods and uses of the present invention relate to the inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) which hydrolyze excess antibiotic residue in the GI tract. Insome embodiments, methods and uses of the present invention relate tothe inventive beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) which maintain a normal intenstinal micrbiota and/orprevents the overgrowth of one or more pathogenic microorganisms in theGI tract of a patient. In various embodiments, the beta-lactamasesand/or pharmaceutical compositions (and/or additional agents) do notsubstantially interfere with plasma levels of a primary antibiotic. Forexample, the beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) of the present invention allow for a patient toreceive a primary antibiotic that might be required for an infection anddo not interfere with the systemic utility of the antibiotic. Rather,the beta-lactamases and/or pharmaceutical compositions (and/oradditional agents) inactivate excess antibiotic that may populate partsof the GI tract and in doing so, prevent the disruption of themicrobiota that is linked to the various disease states describedherein.

In various embodiments, the inventive beta-lactamases and/orpharmaceutical compositions (and/or additional agents) are notsystemically absorbed. In various embodiments, the inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) do not substantially interfere with the activity of systemicallyadministered antibiotics. In various embodiments, the inventivebeta-lactamases and/or pharmaceutical compositions (and/or additionalagents) function to eliminate antibiotics from interfering with themicrobiota of a microbiome (e.g. the gut, including the largeintestine). In some embodiments, the inventive beta-lactamases and/orpharmaceutical compositions (and/or additional agents) do not interferewith the antibiotic absorption from the gut and/or enterohepaticallysufficiently to alter the half-lives of antibiotic circulation. In someembodiments, the inventive beta-lactamases and/or pharmaceuticalcompositions (and/or additional agents) do not interfere with theantibiotic absorption from the gut and/or enterohepatically enough to beclinically important.

In some embodiments, the methods and uses of the present inventioninclude those in which an initial and/or adjunctive therapy isadministered to a patient. Initial and/or adjunctive therapy indicatestherapy that is used to treat CDI and/or C. difficile associated diseaseupon detection of such disease. In some embodiments, the initial and/oradjunctive therapy is one or more of metronidazole, vancomycin,fidaxomicin, rifaximin, fecal bacteriotherapy, probiotic therapy, andantibody therapy, as described herein. In various embodiments, themethods and uses of the present invention include use of the inventivebeta-lactamases as an adjuvant to any of these initial and/or adjunctivetherapies (including co-administration or sequential administration). Invarious embodiments, the methods and uses of the present inventioninclude use of the inventive beta-lactamases in a patient undergoinginitial and/or adjunctive therapies.

In other aspects, the present invention provides the beta-lactamasesand/or pharmaceutical compositions for use in treating anantibiotic-induced adverse effect in the GI tract and/or prevention ortreatment of C. difficile infection (CDI) and/or a C.difficile-associated disease. In other aspects, there are provided usesof the beta-lactamases and/or pharmaceutical compositions for treatingan antibiotic-induced adverse effect in the GI tract and/or preventingor treating a C. difficile infection (CDI) and/or a C.difficile-associated disease. Further, some aspects provide for the useof the disclosed beta-lactamases in the manufacture of a medicament foruse in treating an antibiotic-induced adverse effect in the GI tractand/or prevention or treatment of C. difficile infection (CDI) and/or aC. difficile-associated disease.

In various embodiments, the present invention provides for compositionsand methods that mitigate or prevent the overgrowth of various coliformsin a patient's gut (including coliforms that are virulent and/orantibiotic resistant). In various aspects, the methods and compositionsdescribed herein prevent or diminish secondary infections with resistantorganisms and may, in some embodiments, diminish beta-lactam resistancedevelopment. Further, the methods and compositions described herein mayallow for use of beta-lactam antibiotics which are currently avoided dueto resistance concerns and/or reduce the need for co-administration orco-formulation with one or more beta-lactamase inhibitors (e.g.AUGMENTIN is a mixture of amoxicillin and clavulanic acid).

In some embodiments, the terms “patient” and “subject” are usedinterchangeably. In some embodiments, the subject and/or animal is amammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow,pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee,or baboon. In other embodiments, the subject and/or animal is anon-mammal, such, for example, a zebrafish. In some embodiments, thesubject and/or animal may comprise fluorescently-tagged cells (with e.g.GFP). In some embodiments, the subject and/or animal is a transgenicanimal comprising a fluorescent cell.

In some embodiments, the subject and/or animal is a human. In someembodiments, the human is a pediatric human. In other embodiments, thehuman is an adult human. In other embodiments, the human is a geriatrichuman. In other embodiments, the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 6to about 18 months old, from about 18 to about 36 months old, from about1 to about 5 years old, from about 5 to about 10 years old, from about10 to about 15 years old, from about 15 to about 20 years old, fromabout 20 to about 25 years old, from about 25 to about 30 years old,from about 30 to about 35 years old, from about 35 to about 40 yearsold, from about 40 to about 45 years old, from about 45 to about 50years old, from about 50 to about 55 years old, from about 55 to about60 years old, from about 60 to about 65 years old, from about 65 toabout 70 years old, from about 70 to about 75 years old, from about 75to about 80 years old, from about 80 to about 85 years old, from about85 to about 90 years old, from about 90 to about 95 years old or fromabout 95 to about 100 years old.

In other embodiments, the subject is a non-human animal, and thereforethe invention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

The invention provides kits that can simplify the administration of anyagent described herein. An exemplary kit of the invention comprises anycomposition described herein in unit dosage form. In one embodiment, theunit dosage form is a container, such as a pre-filled syringe, which canbe sterile, containing any agent described herein and a pharmaceuticallyacceptable carrier, diluent, excipient, or vehicle. The kit can furthercomprise a label or printed instructions instructing the use of anyagent described herein. The kit may also include a lid speculum, topicalanesthetic, and a cleaning agent for the administration location. Thekit can also further comprise one or more additional agent describedherein. In one embodiment, the kit comprises a container containing aneffective amount of a composition of the invention and an effectiveamount of another composition, such those described herein.

This invention is further illustrated by the following non-limitingexamples.

EXAMPLES

The following abbreviations are used throughout:

Mutations relative to P1A (based on the Ambler classification) Name Wildtype RS310 (or P1A) D276N IS118 (or P3A) I72S IS222 T160F IS203 R244TIS217 R244T D276K IS215 Q135M IS197 G156R A238T IS235 F33Y D276N IS158F33Y S240P D276N IS230 (or IS181) F33Y A238T D276N IS232 (or IS180) I72SQ135M T160F IS227 (Block 1 mutants) A232G A237S A238G S240D IS191 (Block2 mutants) A232G A237S A238G S240D R244T IS229 A232G A237S A238G S240DD276R IS219 A232G A237S A238G S240D D276K IS221 A232G A237S A238G S240DQ135M IS224 A238T IS233 T243I S266N D276N IS234 (or IS176) A232G A237SA238G S240D D276N IS288 (or P4A)

Example 1: Materials and Methods

Different P1A mutants were made either with site-directed mutagenesisusing SOE or QUIKCHANGE Multi Site-Directed Mutagenesis Kit (Stratagene,CA, USA) or with random mutagenesis using GeneMorph® II RandomMutagenesis Kit (Stratagene, CA, USA). Mutated DNA was transformed intobacteria, either E. coli or B. subtilis, which were screened oncefotaxime (site-directed mutants) or ceftriaxone (random mutants). Thepresence of 8-lactamase in colonies was further confirmed with anitrocefin test and the positive colonies were selected for furtheranalysis. The DNA sequences of the mutant 8-lactamases were confirmed bysequencing.

8-lactamase activity of the mutants was further characterised from E.coli cell lysates and B. subtilis growth supernatant with ampicillin,cefotaxime, ceftriaxone, cefuroxime, ceftazidime, meropenem andimipenem. The activities were determined using only one substrateconcentration of each antibiotic tested. Selection of the antibioticconcentration was done with the antibiotic concentration as high as waspractical, [S]>>Km. Based on these preliminary experiments, moreaccurate kinetic parameters were determined for a couple of mutants fromboth the site-directed and the random mutagenesis research lines. Allthe mutant enzymes selected for more detailed study were expressed inthe B. subtilis host strain RS303. The cells carrying the mutatedproteins were grown in minimal medium over-night after which the cellswere harvested and the supernatant was concentrated in 1:10 with AmiconUltra-4 centrifugal devices, MWCO 10000 (Amicon). Protein concentrationin each culture supernatant was estimated from 12% SDS-PAGE (CriterionXT Bis-Tris, BioRad, CA, USA) using the P1A reference material A18K31 asa standard. Kinetic parameters were measured for cefotaxime, ceftriaxoneand cefuroxime for each selected mutant strain. The site-directedmutants were designed to hydrolyze cefotaxime and random mutants werescreened with ceftriaxone, which made these antibiotics natural choicesfor determining kinetic parameters.

P1A Modifications

A series of mutations in P1A were made, including:

TABLE 1 Mutations on P1A from structure-based design and construction ofcefotaxime degrading P1A derivatives, Block 1 I72S, Q135M, T160F Block 2A232, A237S, A238G, S240D R244T D276R/K Block 1 I72S, Q135M, T160F anddifferent combinations of these

Parallel to the site-directed mutagenesis, random mutagenesis was doneon P1A and P3A with ceftriaxone as a screening antibiotic.

There are also two modification mutants of P1A that were made for thequality control studies: P1A-N293D (IS205, “the deamidation mutant”) andP1A-AMNGK (IS206, “the deletion mutant”). Both of these strains grewequally well as P1A-strain RS310 and the protein production was aboutthe same as well. These mutations did not affect the crystallizabilityof the protein either.

Sequences of selected CTX-M-enzymes were derived from Swiss-Prot proteinsequence data bank and the 3D structures were derived from the ProteinData Bank (PDB). Accession codes for individual sequences and structuresare represented below. In some instances, this data was used to predictstructure functional activity.

Illustrative Swiss-Prot or TrEMBL, GenBank and Protein Data Bank (PDB)accession codes for enzymes used in the study. Swiss-Prot/TrEMBL GenBankPDB CTX-M-9 Q9L5C8_ECOLI (TrEMBL) AF174129 1YLJ CTX-M-14 Q9L5C7_ECOLI(TrEMBL) AF252622 1YLT CTX-M-15 Q9EXV5_ECOLI (TrEMBL) AY044436 —CTX-M-16 Q939K2_KLEPN (TrEMBL) AY029068 1YLW CTX-M-25 Q8KSA6_ECOLI(TrEMBL) AF518567 — CTX-M-44 Q47066 (Swiss-Prot) D37839 1IYO

A Linux workstation and Fedora Core 5 operating system was used as thestudy platform. The sequence alignments and analysis as well as themodelling and 3D structure analysis were performed with Bodil molecularmodelling environment.

Additionally, several other P1A mutants were designed and made. One ofthese mutants was IS288, which has following mutations: A232G, A237S,A238G, A240S and D276N. The four mutations A232G, A237S, A238G and A240Sare a so-called “Block2”-mutation. The fifth mutant, D276N, is the“P3A”-mutation, which alone gives the protein's ability to degradeceftriaxone. The P1A variant IS288 was a result of two rounds ofmutagenesis on the P1A gene and preliminary activity assays of secretedP1A variants. Several P1A variants made by site-directed mutagenesiswere cloned into the B. subtilis production host and produced in shakerflask cultivations. The growth supernatants of these mutant strains wereconcentrated and used to measure each of the strains' ability to degradeselected cephalosporins. IS288 was produced in larger scale and purifiedusing ammonium sulphate precipitation, hydrophobic interactionchromatography and ultra filtration. The study was set up tocharacterize the selected P1A variant IS288 in more detail.

In addition to purifying with HIC, methods were developed to furtherdesalt and characterize the amount and purity of protein in the elutedprotein peak.

Hydrophobic interaction chromatography, i.e. HIC, is a method, where theseparation of bio-molecules is based on interplay between thehydrophobicity of the medium, the nature and composition of the sample,the prevalence and distribution of surface-exposed hydrophobic aminoacid residues, and the type and concentration of salt in the bindingbuffer. The adsorption of protein into the gel matrix is dependent onthe concentration of anti-chaotropic salts such as (NH₄)₂SO₄ and Na₂SO₄;the higher the salt concentration the stronger the adsorption capacityof the HIC medium. The HIC matrix can have different types ofhydrophobic substituents, like butyl, octyl, or phenyl groups. Butylsepharose was selected as the HIC matrix as P1A binds tightly to octylsepharose that it is not properly eluted.

IS288 Purification Procedure:

The first day, a small amount of a frozen IS288 Bacillus subtilis cellculture was streaked on a LB-agar plate containing 10 μg/ml kanamycin.The following morning single cultures were taken from this plate andinoculated into 10×3 ml of minimal growth medium for shaker flaskcultivation. In the afternoon of the same day, a volume resulting in a0.01 starting OD₆₀₀ in the 100 ml overnight (o/n) culture was taken fromthe 3 ml pre-culture. The 100 ml cultures were incubated in a shaker at+37° C. over night. From a couple of 3 ml pre-cultures OD₆₀₀=1 glycerolstocks (10% glycerol) were made. The glycerol stocks were stored at −70°C.

On the third day after ˜16 hours of cultivation, the final OD₆₀₀ of theshaker flask cultivations were measured and cells were centrifuged(Sorvall RC6) with SLA-3000 rotor, 7000 rpm, 10 minutes at +4° C.).Culture supernatants were collected and combined and then filtrated witha 0.2 μm filter. The volume of the filtrate was measured to ˜745 ml.326.86 g of solid (NH₄)₂SO₄ resulting in 70% (NH₄)₂SO₄ saturation wasweighed and added as ˜33 g aliquots into the supernatant whilecontinuously mixing. Once all salt had dissolved, the solution wastransferred to a +4° C. refrigerator.

The following morning, the ammonium sulphate solution was centrifuged(Sorvall RC6) SLA-3000 rotor, 90 minutes, 10500 rpm, ±4° C.,acceleration 9 and deceleration 4). After centrifugation, thesupernatants were combined and filtered through a 0.2 μm sterile filter.72.5 ml of the filtrate was taken for hydrophobic interactionchromatography (HIC). The rest of the filtrate was divided into 17×50 mlaliquots and stored first at ±4° C. and later transferred to −20° C.Moreover, two 1 ml samples of the filtrate were taken and stored atfirst at ±4° C. and later transferred to −20° C.

The (NH₄)₂SO₄ concentration of the ammonium sulphate filtrate wasestimated to be ˜2.7 M and it was diluted so that the concentration of(NH₄)₂SO₄ decreased to 2 M with 190 mM Na—H-Phosphate buffer, pH 6.8.The filtrate was subjected to HIC in a 5 ml HiTrap Butyl FF columnequilibrated with 50 mM Na—H-Phosphate, pH 6.9, 2 M (NH₄)₂SO₄. A totalof 50 ml of sample was pumped through the column and the flow-throughwas collected as 5 ml fractions. Unbound sample was washed out with 50mM Na—H-Phosphate, pH 6.9, 2 M (NH₄)₂SO₄. Bound IS288 was eluted withdecreasing (NH₄)₂SO₄ concentration using step gradient and 50 mMNa—H-Phosphate, pH 6.8. In the first run, elution fractions were 1 ml.Fractions containing the eluted protein were combined in three differentsets; C3-C8, C9-D1 and D2-D8. Flow-through fractions and the rest of theelution fractions were stored at ±4° C. Samples were taken fromflow-through fractions B1 and B8 as well as the pooled protein fractionsand stored at ±4° C.

The HIC step was repeated two times for different sample volumes, namely65 ml and 125 ml of equilibrated ammonium sulphate filtrate. These laterHIC runs were somewhat modified from the first run. The wash volume forunbound sample was 3 and 4 CV, the volume of flow-through fractions wasincreased to 10 ml and the volume of the elution fractions was increasedto 1.5 ml. No other modifications were made to the running conditions.

Pooled protein fractions as well as flow-through fractions of B1 and B8of the first run were ultra filtrated and concentrated using AmiconUltra-4 concentrators with 10000 NMWL. The samples were centrifuged sixtimes and after each centrifugation, except the last one, the rest ofthe sample or buffer was added into the retentate so as to gain 4 mlvolume. After the sixth concentration step, retentates were collected,measured and stored at ±4° C. Samples from succeeding HIC runs, as wellas one more flow through fraction from the first run were ultrafiltrated and concentrated with Amicon Ultra-15 with 10000 NMWL and onlyfour centrifugation rounds were needed. These samples were also storedat ±4° C. Ammonium sulphate filtrates were ultra filtrated andconcentrated, and flow through fractions were once more concentratedwith MultiScreen filter plates with Ultracell-10 membrane, 10000 NMWL.Protein concentration of the flow through samples was measured beforethis last concentration step whereas no protein concentration wasmeasured for ammonium sulphate filtrates.

The protein concentration of the samples was measured with BCA ProteinAssay kit. Based on the elution peak obtained from Äkta, eluted proteinfractions were diluted 1:20 and 1:40 while the flow-through fractionswere used as such (1:1 dilution) or diluted in half (1:2 dilution).

Finally, samples from all three purifications were run on a Criterion XT12% Bis-Tris SDS-PAGE using MOPS running buffer to optimally separateproteins of 6 66 kDa in size. The maximum amount of sample was added forammonium sulphate filtrates and all flow through fractions except forB1₁₂₁₁₀₈ for which the protein content was determined to be 0.21 μg/μlin the 33× concentrated sample. The amount of protein peak fractionspipetted was calculated so that 0.5 μg of protein would be loaded on thegel.

A purified protein solution of IS288 was used to determine the activityof IS288 per mass unit. The stability of IS288 in storage conditions wasexamined to find out if the protein is degraded or by some other waymodified during storage. The stability of the protein at the target sitematrix was also determined. The results from the stability assay intarget site matrix were combined with the results from the activityassay to estimate the time it takes for IS288 to eliminate antibioticresidues in the small intestine.

In addition to getting more detailed information on P1A variant IS288,the characterization scheme created during the study was used in thecharacterization of other subsequent P1A variants.

Method to Study Production Levels of P1A Variant IS288

During the Bacillus subtilis shaker flask cultivation, samples weretaken at various time points to evaluate the production level of IS288.The OD₆₀₀ value was measured for each of the time points as well to seehow the cells were growing. The amount of produced protein was estimatedon SDS-PAGE.

Method to Study Enzyme Efficacy and Antibiotic Spectrum at Different pHLevels

The antibiotics used in this study were a selection of, e.g.,intravenously administered cephalosporins. The concentrations ofselected antibiotics were, without wishing to be bound by theory, set toas close to the in literature reported concentrations in bile orduodenum as possible. This experiment was also performed at varying pHs.

Method to Study Enzyme Stability in Human Ileal Chyme

Different amounts of purified enzyme were incubated in human ileal chymeat the appropriate temperature for a set period of time. Samples weretaken at different time points. The degradation pattern of IS288 wasanalysed with SDS-PAGE and the activity of IS288 was analysed usingnitrocefin as a substrate.

Compounds and Bacterial Strain: Ampicillin, Ceftriaxone, Ceftazidime,Meropenem, Cefepime, Cefazolin, Amp/Sulbactam, Cefoperazone, Cefotaxime,and Cefuroxime were purchased from commercial sources and stored asfrozen stocks of 15 mg/mL and 10 mg/mL, respectively. P1A was suppliedby Synthetic Biologics as a stock concentration of 32 mg/mL and storedat −80° C. Escherichia coli ATCC 25922 was obtained from American TypeCulture Collection (ATCC, Manassas, Va.).

Determination of the Effective Inhibitory Concentration in the Absenceand Presence of P1A (RS310), P3A (IS118), and P4A (IS288): The effectiveinhibitory concentrations (EC50, 90) of ampicillin, ceftriaxone,ceftazidime, meropenem, cefepime, cefazolin, amp/sulbactam,cefoperazone, cefotaxime, and cefuroxime were determined by microbrothdilution analysis according to the CLSI guidelines (M07-A9: Methods forDilution Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically; Approved Standard Ninth Edition). Escherichia coli strain25922 was cultured from glycerol stocks to logarithmic growth in cationadjusted Mueller-Hinton broth at 37° C. in an atmospheric environmentand diluted in cation adjusted Mueller-Hinton broth to achieve astarting in well concentration of 5×105 CFU/mL. Serial 2-fold dilutionof antimicrobial compounds were prepared in cation adjustedMueller-Hinton broth to achieve final in well concentration of 128 μg/mLto 0.13 μg/mL for ampicillin and 4 μg/mL 4 ng/mL in the absence orpresence of serial 10-fold dilutions of P1A, P3A, or P4A β-lactamasestandard, 5 μg/mL to 50 ng/mL for ampicillin and 50 μg/mL to 500 ng/mLfor ceftriaxone, ceftazidime, meropenem, cefepime, cefazolin,amp/sulbactam, cefoperazone, cefotaxime, and cefuroxime. Antibioticswere either added to the P1A and incubated for 30 min at 37° C.(pre-incubation) prior to addition of bacteria, or added to the platesfollowing the addition of P1A, P3A, P4A, and bacteria (simultaneous).All concentrations and combinations were evaluated in triplicate in a96-well plate format that contained media only, compound plus media only(colorimetric control), and untreated bacterial controls. Followingovernight incubation, the OD625 of the plates was measured in aSpectramax 384 plate reader and the data were imported into a customizedExcel spreadsheet for the determination of the effective concentrationthat inhibited bacterial growth by 50%, 90% and 99% (EC50, 90, 99) bylinear regression analysis.

Example 2: Results of Mutagenesis

Mutation design was based on, inter alia, structural data (e.g. crystalstructure data, homolog models, etc.) of the following: P1A crystalstructure (Knox and Moews, J. Mol Biol., 220, 435-455 (1991)), CTX-M-44(1BZA (Ibuka et al Journal of Molecular Biology Volume 285, Issue 52079-2087 (1999), 1IYS (Ibuka et al. Biochemistry, 2003, 42 (36):10634-43), 1IYO, 1IYP and 1IYQ (Shimamura et al 2002 J. Biol. Chem.277:46601-08), Proteus vulgaris K1 (1HZO, Nugaka et al. J Mol Biol. 2002Mar. 15; 317(1):109-17) and Proteus penneri HugA (Liassine et al.Antimicrob Agents Chemother. 2002 January; 46(1):216-9. 2002), andreviewed in Bonnet, Antimicrob. Agents Chemother 48(1): 1-14 (2004) (forCTM-X), the contents of all of these documents are hereby incorporatedby reference in their entirety). In some embodiments, the presentmutations are informed by analysis of structural data (e.g. crystalstructure data, homolog models, etc.) of any one of the followingβ-lactamases: P1A, P2A, P3A, CTX-M-3, CTX-M-4, CTX-M-5, CTX-M-9,CTX-M-10, CTX-M-14, CTX-M-15, CTX-M-16, CTX-M-18, CTX-M-19, CTX-M-25,CTX-M-26, CTX-M-27, CTX-M-32, CTX-M-44, CTX-M-45, and CTX-M-54. Suchinformation is available to one skilled in the art at known databases,for example, Swiss-Prot Protein Sequence Data Bank, NCBI, and PDB.

Results from site-directed and random mutagenesis are summarized inTables 2 and 3, respectively. Enzymatic activity for selected mutants ispresented in Table 4 and kinetic parameters are represented in Table 5.Kinetic parameters for previous enzymes P1A, P3A and P2A are alsoincluded in that table.

Mutation Block 1. I72S, Q135M and T160F

The first block of mutations including I72S, Q135M and T160F wereintroduced in mutagenic primers in one reaction using The QUIKCHANGEMulti Site-Directed mutagenesis kit (for short later called Multi kit),Stratagene. Plasmid carrying the penP gene was used as template in themutagenesis reaction. Mutant single-stranded plasmids were transformedinto E. coli XL10Gold and selected with kanamycin/chloramphenicol.Competent XL10 Gold cells are provided with the Multi kit. Colonies(10-30) were picked and tested with nitrocefin to verify 8-lactamaseactivity. Part of the colony was used as template in PCR and theresulting fragment, if clean, was sent to DNA sequencing. The rest ofthe colony was cultivated in Luria supplemented with appropriateantibiotic, overnight, at 37° C., and a part of the culture was storedin glycerol (10%, final), another part is used for plasmid isolation.After the desired mutations were confirmed with DNA sequencing, themutant clones were characterized in more detail. Periplasmic lysateswere prepared and analyzed in SDS PAGE and the kinetics of cefotaximehydrolysis were measured spectrophotometrically.

Mutation Block 2. A232G, A237S, A238G and S240D

The target amino acids of the B3 β-strand of P1A are so closely situatedto each other that the Multi-Site protocol was not suitable, at least inone round. Instead, all four amino acids were exchanged using the SOEtechnique. The region encompassing the exchanges contained 24nucleotides (8 amino acid codons) and was included in the overlapextension part of two PCR primers. The mutated penP gene was firstPCR'ed in two parts, using primers A+B and C+D (FIG. 7). The two PCRproducts were then combined and amplified using primers A and D,complementary to the 5′ and 3′ parts of the penP gene. Cloning siteswere included in primers A and D. Once the construct was ready, furthermutations were added using the Multi kit.

Further Mutations, R244T and D276R(K)

These mutations were added using the Multi-Site kit technique and thepreviously made mutant plasmids as templates. A list of the constructedmutations is shown in Table 1. The number of mutation combinations wasincreased easily and consequently, the characterization was done withmore robust methods than measuring the cefotaxime degradation kineticsof individual clones. Primary characterization was the determination ofthe cefotaxime MIC.

TABLE 1 Constructed mutations and other possible combinations CloneTechnique Amino acid replacements Block 1 Multi kit I72S, Q135M, T160FMulti kit I72S, T160F Multi kit I72S, Q135M Multi kit I72S Multi kitQ135M Multi kit T160F Block 2 SOE A232G, A237S, A238G, S240D SOE + Multikit A232G, A237S, A238G, S240D + R244T SOE + Multi kit A232G, A237S,A238G, S240D + D276R(K) SOE + Multi kit A232G, A237S, A238G, S240D +R244T + D276R(K) SOE + Multi kit A232G, A237S, A238G, S240D + I72S,Q135M, T160F SOE + Multi kit A232G, A237S, A238G, S240D + I72S, Q135M,T160F + R244T SOE + Multi kit A232G, A237S, A238G, S240D + I72S, Q135M,T160F + D276R(K) SOE + Multi kit A232G, A237S, A238G, S240D + I72S,Q135M, T160F + R244T + D276R(K) Multi kit R244T Multi kit D276R(K) Multikit R244T + D276R(K) Multi kit I72S, Q135M, T160F + R244T Multi kitI72S, Q135M, T160F + D276R(K) Multi kit I72S, Q135M, T160F + R244T +D276R(K)

TABLE 2 P1A mutants made using site-directed mutagenesis. B. subtilisand E. coli strain numbers, mutations and plasmid names are marked.Plasmid B. subtilis name in E. coli strain Amino acid replacements B.subtilis strain IS191 Block 2 = A232G, A237S, pCTX-42 IS189 A238G, S240DIS197 Q135M pIS197 IS193 IS215 R244T + D276K pIS215 IS207 IS217 R244TpIS217 IS108 IS219 Block 2 + D276T pIS219 IS209 IS221 Block 2 + D276KpIS221 IS210 IS222 I72S pIS222 IS212 IS224 Block 2 + Q135M pIS224 IS214IS227 Block 1 = I72S, Q135M, pRSH227 SOE T160F IS229 Block 2 + R244TpRSH229 SOE

TABLE 3 P1A mutants generated with random mutagenesis. B. subtilis andE. coli strain numbers, mutations and plasmid names are marked. PlasmidB. subtilis name in E. coli strain Amino acid replacements B. subtilisstrain IS158 F33Y, D276N pRSH158 IS230 F33Y, S240P, D276N pRSH230 IS181IS232 F33Y, A238T, D276N pRSH232 IS180 IS234 R55R, A123A, T243I, pRSH234IS176 S266N, D276N

TABLE 4 Preliminary enzymatic activities of P1A mutants measured from B.subtilis supernatants. B. subtilis strain Amino acid replacements AMPCTX CRO CXM CAZ RS310 P1A strain 1.00 1.00 1.00 1.00 1.00 IS191 A232GA237S 0.16 2.17 2.91 3.22 1.28 A238G S240D IS197 Q135M 0.15 1.32 1.091.83 0.55 IS203 T160F 0.01 0.01 0.00 0.00 0.13 IS215 R244T D276K 0.440.50 0.93 0.81 0.26 IS217 R244T 0.22 0.00 0.00 0.05 0.11 IS219 A232GA237S A238G 0.05 7.22 15.99 7.67 0.27 S240D D276R IS221 A232G A237SA238G 0.06 9.39 15.95 9.87 0.38 S240D D276K IS222 I72S 0.71 0.00 0.150.18 0.00 IS224 A232G A237S A238G 0.02 4.74 5.18 6.37 1.24 S240D Q135MIS230 F33Y S240P D276N 0.02 8.07 9.26 4.76 8.98 IS232 F33Y A238T D276N0.08 12.34 9.14 5.71 26.73 IS233 A238T 0.07 4.17 5.25 4.76 12.70 IS234T243I S266N D276N 0.57 6.04 11.03 6.86 11.23 IS235 G156R A238T 0.03 1.662.09 1.34 5.42 Values are represented as multiples of P1A. AMP =ampicillin, CTX = cefotaxime, CRO = ceftriaxone, CXM = cefuroxime andCAZ = ceftazidime, P1A values used for the calculations are means of twodifferent experiments.

TABLE 5 Kinetic parameters measured for the P1A mutant strains. Kineticparameters for IS230 degrading cefuroxime are not reliable as theoriginal curve did not obey Michaelis-Menten kinetics. Moreover, itshould be noted that kinetic parameters for IS219 and IS221 are near oneanother except when cefotaxime is degraded; when the V_(max) for IS219is increased, it is decreased for IS221. On the other hand, the affinityof IS219 towards cefotaxime has not increased with the same amount asIS221's. Ceftriaxone Cefotaxime Cefuroxime V_(max) K_(m) k_(cat)k_(cat)/Km V_(max) K_(m) k_(cat) k_(cat)/K_(m) V_(max) K_(m) k_(cat)k_(cat)/K_(m) (nmol/s) (mM) (1/s) (M⁻¹ * s⁻¹) (nmol/s) (mM) (1/s) (M⁻¹ *s⁻¹) (nmol/s) (mM) (1/s) (M⁻¹ * s⁻¹) P1A 2.81 179 25 1.41 * 10⁵ 1.18 23216  6.8 * 10⁴ 1.25 107 23 2.11 * 10⁵ (A18K31) IS219 0.37 47 171 3.62 *10⁶ 1.51 164 60 3.68 * 10⁵ 0.94 140 126 8.98 * 10⁵ IS221 0.30 30 1033.46 * 10⁶ 0.43 66 43 6.47 * 10⁵ 0.90 108 89 8.22 * 10⁵ IS227 Low LowLow Low Low Low Low Low IS229 Low Low Low Low 3.10 574 136 2.37 * 10⁵P3A⁽¹ 0.25 55 54 9.89 * 10⁵ 0.24 230 17  7.3 * 10⁴ ND ND ND ND IS1581.27 33 45 1.36 * 10⁶ 0.57 144 20 1.41 * 10⁵ ND ND ND ND IS232 0.47 4 276.25 * 10⁶ 0.31 14 23 1.69 * 10⁶ low low low low (IS180) IS234 1.09 3753 1.45 * 10⁶ 0.61 33 30 8.88 * 10⁵ 0.77 17 38 2.19 * 10⁶ (IS176) IS2300.66 18 32 1.80 * 10⁶ 0.47 21 23 1.09 * 10⁶ 0.50 7 24 3.27 * 10⁶ (IS181)P2A⁽¹ 68 95 1.40 * 10⁶ 0.79 66 479 7.28 * 10⁶ 0.37 27 221 7.99 * 10⁶ Low= not enough activity for measurements; ND or empty cell = not done.

TABLE 6 V_(max) and K_(m) values of selected mutants represented asrelative values of P1A activities. If K_(m) <1.0, the mutant's affinitytowards its ligand increased. If K_(m) >1.0, the affinity decreased. Theopposite is true for k_(cat) and V_(max). Ceftriaxone CefotaximeCefuroxime V_(max) K_(m) k_(cat) k_(cat)/K_(m) V_(max) K_(m) k_(cat)k_(cat)/K_(m) V_(max) K_(m) k_(cat) k_(cat)/K_(m) P1A 1.00 1.00 1.001.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 (A18K31) IS219 0.13 0.266.76 25.64 1.29 0.71 3.81 5.39 0.75 1.31 5.56 4.25 IS221 0.11 0.17 4.0924.46 0.37 0.29 2.70 9.48 0.71 1.01 3.93 3.89 IS227 low low low low lowlow low low — — — — IS229 low low low low 2.64 2.47 8.58 3.47 — — — —P3A 0.09 0.31 2.15 7.00 0.20 0.99 1.07 1.07 — — — — IS158 0.45 0.19 1.809.63 0.48 0.62 1.28 2.06 — — — — IS232 0.17 0.02 1.08 44.25 0.26 0.061.44 24.70 — — — — (IS180) IS234 0.39 0.20 2.10 10.26 0.52 0.14 1.8713.00 0.61 0.16 1.66 10.38 (IS176) IS230 0.24 0.10 1.27 12.70 0.40 0.091.45 15.99 0.40 0.07 1.08 15.48 (IS181) P2A — 0.38 3.76 9.87 0.67 0.2830.18  106.55 0.29 0.25 9.78 37.81 Low = low activity; — = not done.Enzymatic Activity and Kinetic Parameters

From the kinetic parameters measured for P1A against cefotaxime,ceftriaxone and cefuroxime, without wishing to be bound by theory, itwas deduced that P1A has a low intrinsic cephalosporinase activity. Ingeneral, the mutants' affinity towards ligands has increased (i.e.decreased Km). V_(max) increased and the affinity decreased (Kmincreased) in the 18229 (Block 2+R244T) mutant (Table 5). For 18229, thek_(cat) increased the most.

The random mutagenesis screen with ceftriaxone preferred mutants withlow Km (high affinity) towards the antibiotic.

Decrease in Km by subsequent mutation rounds was seen in the mutationseries P3A→IS158→IS232; IS232 had extremely high affinity towardsceftriaxone. The V_(max) for ceftriaxone did not decrease sequentiallybeing the highest for IS158 but k_(cat), nevertheless, decreased throughthe mutation rounds. The first two mutants of this round did not havecefotaximase activity but 18232 with additional A238T had cefotaximaseactivity.

Site-directed Block2 (IS191) mutations alone did not markedly increaseP1A-derivative's activity towards cephalosporins but the activitytowards ampicillin was reduced 20 fold (Table 4). The cephalosporinaseactivity of Block2±D276R/K (IS219/IS221) was considerably better thanthat of IS191's. The kinetic parameters of the IS219 and IS221site-directed mutants revealed that the site-directed mutagenesispreferably increased the enzyme's affinity towards the targetantibiotics.

Stability Assay/Glucose Stress Test

To test the stability of mutants in B. subtilis growth conditions, ashaker flask cultivation with RS310, 1S219 and IS232 was performed. Thecultivation was made in two different glucose concentrations of 5 g/land 10 g/l.

P1A (RS310) and the two mutants IS219 and IS232 were cultivated inshaker flasks (100 ml) in minimal medium with 5 or 10 g/l glucose. Thecondition of the cells was monitored with microscope, the glucoseconsumption measured from 5 g/l growth and samples from the culturesupernatants (concentrated and non-concentrated) were run on 12%Bis-Tris SDS-PAGE (Criterion, BioRad) (FIGS. 1 and 2). The ODs of thecell cultures were also monitored.

In 10 g/l glucose cultures, P1A and IS232 were produced in comparableamounts but the production of IS219 was lower (lanes 2-4, FIG. 1).During concentration, the IS219 yield was further reduced (lanes 6, 9and 16, FIG. 1). A faint band ˜29 kDa can be seen on lane 16 in FIG. 1.P1A and IS232, again, could be concentrated (lanes 5, 7, 8, 10, 15 and16 FIG. 1).

In the presence of 5 g/l glucose, P1A strain RS310 started growingslower than the other two strains. However, by the end of thecultivation the amount of P1A had superseded the amount of the otherproteins (FIG. 2). The mutant strain IS219 started to produce roughly atthe same velocity as the strain IS232 but by 10 hours of cultivation itsproduction had started to lag behind IS232 and its amount was decreasedover time. All glucose was consumed by 9.5 hours of cultivation both inIS219 and IS232 cultivations and from that point on it is assumed thatthe amount of proteases started to increase. Because RS310 grew slower,it still had some glucose left at 11 hours of cultivation but also RS310had consumed all glucose by the following morning. Because no sampleswere taken from RS310 during the night, the exact time-point at whichRS310 had used all its glucose and started to produce proteases cannotbe stated. By 27.5 hours of cultivation the amount of P1A had increasedbut the amount of both IS219 and IS232 had decreased from the previousday. From the study, without wishing to be bound by theory, it can bededuced that IS232 tolerates proteases better than IS219, which does nottolerate them at all.

Based both on 10 g/l glucose and 5 g/l glucose cultivations, withoutwishing to be bound by theory, it can be said that modifications to P1Areduce the protein's protease tolerance. Glucose deprivation lead to theappearance of degradation products in the case of IS219 and IS232 andthe amount of degradation products was increased after all glucose wasconsumed. IS219 protein was more sensitive to degradation as its amountdecreased more than that of IS232. Without wishing to be bound bytheory, this may indicate either lower secretability or greatersensitivity to Bacillus proteases, or both. In conclusion, production ofIS219 was not possible using the same growth conditions as for RS310 andIS232. The glucose stress test also demonstrated that this test can beused to estimate the protease tolerance of the generated mutants.

With the glucose stress test it was demonstrated that Block2+D276R=IS219(and most likely Block2+D276K=IS221) mutation decreases the enzyme'sprotease tolerance. Most likely, without wishing to be bound by theory,the instability is due to D276R mutation, which creates a proteasecleavage site on the surface of the protein.

A D276N mutation on IS191, which would result in a Block2+D276N mutantwas made. Without wishing to be bound by theory, an aspartic acid toasparagine mutation is expected to increase the ceftriaxone (and may becefotaxime) activity of IS191 but at the same time keep the enzyme moreresistant to proteases than IS219 (and IS221) is. R244T mutation couldbe combined with Block2 and D276N to see whether we can increase theV_(max) (and k_(cat)) with this mutation. R244T mutation could also becombined with IS219 and IS221.

An activity assay (Table 4) R244T (IS217) mutation abolished nearly allenzymatic activity of P1A. Some of the activity was gained with a doublemutant R244T+D276K (IS215), but not to the level of the wild typeenzyme. Thus, R244T mutation alone cannot provide P1A withcephalosporinase activity.

Based on kinetic data (Table 5) Block1 mutation (I72S/Q135M/T160F,IS227) did not improve P1A's ability to hydrolyze cephalosporins. Inother activity assays neither did Q135M or T160F alone increase P1A'sability to hydrolyze cephalosporins, instead, both single mutationsabolished nearly all P1A's enzymatic activity, even towards ampicillin.There was, however, a Block2+Q135M-mutant (IS224), which had an activitytowards cefotaxime and ceftriaxone between Block2 (IS191) andBlock2+D276R/K (IS219/IS221) mutants. Without wishing to be bound bytheory, Block1 may “loosen” the Ω-loop where the catalytic Glu166 islocated. T160F mutation is quite a large modification in the core of aprotein's structure. It might disrupt the structure totally or at leastincapacitate the protein from functioning properly. To avoid thispossibility, two compensatory mutations were added to accompany T160Fmutation in P1A, namely I72S and Q135M and this combination of mutationswas called “Block1”.

Example 3: Results from Production and Purification of P1A Mutant IS288

The results of the purification are reported as elution peak figures(FIGS. 3, 4, and 5) and peak tables, as protein concentration and asSDS-PAGE picture(s) (FIG. 6).

Chromatograms from Hydrophobic Interaction Chromatography

Hydrophobic interaction chromatography (HIC) was run on three separateoccasions with differing parameters. In the first run (16 Oct. 2008,FIG. 3) the sample volume was 50 ml, volume of flow through fractions 5ml, wash volume for the unbound sample 2 CV, and the volume of elutedfractions 1.0 ml. In the following HIC run (4 Nov. 2008, FIG. 4) thesample volume was increased to 65 ml, flow-through fraction volume to 10ml, and wash volume for unbound sample to 3 CV and fraction size to 1.5ml. In the last run (12 Nov. 2008, FIG. 5) only the sample volume andwash volume for unbound sample differed from the preceding run being 125ml and 4 CV, respectively.

All the graphs of HIC runs (FIGS. 3, 4, and 5) represent the sameoverall form. The flow through fraction gave constant absorbance ˜700mAU at 280 nm throughout the sample application while during washing ofunbound sample, the absorbance at 280 nm dropped to zero rising againwhen the bound protein was eluted. The conductivity of the sample wasslightly lower in samples applied in FIG. 3 and FIG. 4 than in runningbuffer A containing 2 M (NH₄)₂SO₄ while in the run on Dec. 11, 2008 itstayed the same as in the A-buffer. In the two first runs the (NH₄)₂SO₄concentration of the ammonium sulphate filtrate was estimated as 2.7 Mand in the last run 2.6 M. It seems that the 2.6 M estimation was closerto the actual (NH₄)₂SO₄ concentration of the ammonium sulphate filtrateas the conductivity of the out coming solution did not drop duringsample application (FIG. 5).

The elution that started right after the elution buffer (buffer B) wasapplied on the column. This indicated that the protein in the sample wasbarely bound to the column as it eluted so early in the gradient. Thus,it was vital that the conductivity of the sample never got less than˜240 mS/cm.

The bound protein eluted as a typical elution peak with steep ascendingslope and a gentle descending slope giving slightly asymmetrical peak.The size of the peak was largest in the first run, −18 ml (18×1 mlfractions) (FIG. 3). This peak was collected in three sets: first of 6ml (fractions C3-C8), second of 5 ml (fractions C9-D1) and third of 7 ml(fractions D2-D8) as there was a “shoulder” in the elution peak betweenfractions C8 and C9 and it was considered that the last 7 fractionscould be too diluted for concentration. In the two subsequent HIC runs(4 Nov. 2008 and 12 Nov. 2008, FIGS. 4 and 5, respectively) the elutionpeak was 13.5 ml (9×1.5 ml fractions, 4 Nov. 2008) and 12 ml (8×1.5 mlfractions, Dec. 11, 2008) and the whole peak was pooled to one fraction.

In Table 7, the statistical values for A₂₈₀ graphs of the three HIC runsare given. The area of the peak was used to estimate the protein contentof that sample and it was used in estimating the dilution factor fordifferent samples in the BCA assay. The calculations required thetheoretical extinction coefficient of the protein, which was calculatedusing ProtParamTool at ExPASy web service. For IS288, this coefficientwas 25440 M⁻¹ cm⁻¹ and gave A280 0.869 for 0.1% (=1 g/l) solution.

Using the proportion above, the amounts of protein in eluted proteinsamples and the known volumes of these samples, the protein amounteluted from the column was calculated. The formula used was:((mAU*ml)*10^−3/V_(fraction))/0.869*V_(fraction)→(mAU*ml)*10^−3/0.869,which resulted in: 3.36 mg protein 161008, 2.95 mg 041108, and 3.60 mg121108.

TABLE 7 Peak table from three separate HIC runs The date of the run,peak name, retention time i.e. the volume at which the peak reaches itstop, area of the peak, and the maximum height of the peak are given. Thearea of the peak was used to estimate the amount of eluted protein inthe elution fraction. Estimated Retention Area Height protein amountDate Peak name (ml) (mAU*ml) (mAU) (mg) 16 Oct. 2008 Flow through 84.4844814.9666 816.803 fraction Wash unbound 86.54 4640.2506 659.512 sampleEluted sample 93.08 2923.2825 814.968 3.36 4 Nov. 2008 Flow through27.94 42450.2080 673.843 fraction Eluted sample 87.01 2562.0783 953.2772.95 12 Nov. 2008 Flow-through 23.40 78004.2505 658.094 fraction Elutedprotein 152.03 3126.5816 1214.232 3.60Ultra Filtration and Concentration of Flow-Through and Eluted ProteinFractions

From each of the purifications one, two or three of the flow-throughfractions were selected for concentration as well as all fractionscontaining the eluted protein peak. The selected samples are representedin Table 8. Also represented in the table are the starting volume ofthat sample as well as final volume and concentration factor.

TABLE 8 Samples selected for ultra filtration and concentration withAmicon Ultra-4, 10000 NMWL The original volume of the sample and thefinal volume of the sample after concentration as well as theconcentration factor are presented. The concentration factor wascalculated by dividing the original volume by the final volume of thesample after concentration. Original Final Date of volume volume Conc.purification Selected fractions (ml) (μl) factor 16^(th) Oct. 2008 B1(FT) 5 750 6.7 X B7 (FT) 4.5 1000 4.5 X B8 (FT) 5 750 6.7 X C3-C8(Protein Peak) 6 900 6.7 X C8-D1 (Protein Peak) 5 900 5.6 X D2-D8(Protein Peak) 7 900 7.8X  4^(th) Nov. 2008 A2 (FT) 9.5 750 12.7 X  A7(FT) 9.5 750 12.7 X  B2-B10 (Protein Peak) 13 700 18.6 X  12^(th) Nov.2008 B1 (FT) 9.5 900 10.6 X  B8-C3 (Protein Peak) 11 1200 9.2 X

Flow-through fractions selected for the ultra filtration were from thebeginning (61/161,008 and A2₀₄₁₁₀₈) and from the end of sampleapplication (B7₁₆₁₀₀₈, B8₁₆₁₀₀₈, A7₀₄₁₁₀₈ and B1₁₂₁₁₀₈). These sampleswere analysed in order to see if the capacity of the column was exceededwith that particular sample amount.

Moreover, ammonium sulphate filtrates for each of the purifications wereultra filtrated and concentrated and the once concentrated flow throughfractions were still further concentrated with MultiScreen plates withUltracell-10 membrane, 10000 NMWL. The results of that concentration arepresented in Table 9.

TABLE 9 Samples selected for ultra filtration and concentration withMultiScreen Filter Plates with Ultracell-10 membrane, 10000 NMWL Boththe original volume and the volume of the sample after concentration areindicated. Concentration factor was calculated by dividing the originalsample volume with the final sample volume. Original Final Date ofvolume volume Conc. purification Selected fractions (ml) (μl) factor16^(th) Oct. 2008 AS-sup 161008 500 420 1.2 X B1 (FT) 400 125 3.2 X B7(FT) 1200 300 4.0 X B8 (FT) 380 115 3.3 X 4^(th) Nov. 2008 AS-sup 041108500 440 1.1 X A2 (FT) 360 115 3.1 X A7 (FT) 700 265 2.1 X 12^(th) Nov.2008 AS-sup 121108 500 455 1.1 X B1 (FT) 430 165 2.6 X

The flow through fractions of the HIC runs were, thus, concentrated by afactor of 16 or more. The final concentration factors of the flowthrough fractions are listed in Table 10.

TABLE 10 Final concentration factors of flow through samples The finalconcentration factors were calculated by multiplying the concentrationfactor of the first concentration with the concentration factor of thesecond concentration. Date of Selected Conc. purification fractionsfactor 16^(th) Oct. 2008 B1 (FT) 20.0 X B7 (FT) 16.7 X B8 (FT) 21.2 X4^(th) Nov. 2008 A2 (FT) 39.1 X A7 (FT) 35.8 X 12^(th) Nov. 2008 B1 (FT)33.0 XBCA Assay Results

Results from the BCA assay are presented in Table 11. The proteinconcentration of the flow through fractions was measured before the lastconcentration step with MultiScreen plates with Ultracell-10 membrane.

TABLE 11 Protein concentrations of HIC flow-through and peak fractionsThe protein concentration was determined from two replicates of twodifferent dilutions of a given sample. The final volume of the samplewas measured after concentration. Protein Amount of concentration Finalvolume protein in in the sample of the sample the sample Sample name(mg/ml) (ml) (mg) B1₁₆₁₀₀₈ 0.02 0.75 0.015 B8₁₆₁₀₀₈ 0.01 0.75 0.008C3-C8₁₆₁₀₀₈ 9.59 0.9 8.63 C9-D1₁₆₁₀₀₈ 0.10 0.9 0.09 D2-D8₁₆₁₀₀₈ 0.72 0.90.65 Total protein amount 9.37 in fractions C3-D8 A2₀₄₁₁₀₈ 0.00 0.750.00 B2-B10₀₄₁₁₀₈ 9.45 0.7 6.62 B1₁₂₁₁₀₈ 0.08 0.9 0.07 B8-C3₁₂₁₁₀₈ 7.561.2 9.07

Chromatograms from HIC runs gave rather high, 700 mAU, A280 valuesthroughout sample application for the out coming solution. The elutedprotein peaks contained sufficient amounts of protein. They were nearthe presumed binding capacity of the column, 10 mg. The area of the peakfrom the Akta gave three times lower estimates for protein amount thanthe BCA assay.

SDS-PAGE

The maximum volume (17.5 μl) of sample was loaded on the SDS-PAGE (FIG.6) for ammonium sulphate filtrates and flow through fractions, exceptfor B1₁₂₁₁₀₈ for which 6.5 μl was loaded. The volume of the samples fromprotein peaks was calculated so that there would be 0.5 pg of proteinper lane and assumed that all protein in the sample would be P1Aderivative IS288. The SDS-PAGE is represented in FIG. 6.

Ammonium sulphate filtrate samples all contain a double band near 30 kDa(MW of IS288 is 29288 Da, and of P1A 29273 Da). The same double bandaccompanied by a slightly smaller extra band can be clearly seen in flowthrough fraction B1₁₂₁₁₀₈ and somewhat more faintly in flow throughfraction A7₀₄₁₁₀₈. On the other hand, both of these extra bands havebeen removed from eluted protein peaks. Some minor impurities can beseen on the last pooled fractions of purification D2-D8 161008, when 10times the intended amount of protein was loaded. It can be concludedthat the purified protein was the major protein form and that bymolecular weight analysis it was the P1A variant IS288.

Example 4: Test of a Microbiological Screening Assay for Beta-LactamaseActivity

The 50% effective inhibitory concentration (EC₅₀) of ampicillin andceftriaxone against Escherichia coli was determined in the absence andpresence of P1A β-lactamase. For the purposes of these studies, thestated effective concentration was the initial concentration ofampicillin or ceftriaxone added at the beginning of the experiment, andnot a concentration measured during the course of the experiment or atthe end of the incubation period. The results of the analysis arepresented in Tables 12-14.

Initial evaluation utilized P1A at concentrations of 0.5 ng/mL to 5μg/mL and ampicillin or ceftriaxone at concentrations of 0.13 μg/mL to128 μg/mL. Addition of the lowest concentration of P1A (0.5 ng/mL)shifted the EC₅₀ of ampicillin from 16 μg/mL to greater than the highesttest concentration of 128 μg/mL when added simultaneously and to 64μg/mL when P1A was pre-incubated with ampicillin prior to addition ofthe bacteria (Table 12). Using the same concentrations of P1A with 0.13μg/mL to 128 μg/mL of ceftriaxone, it was observed that ceftriaxone wascompletely inhibitory at the lowest concentration (EC₉₉<0.13 μg/mL) andthe EC₅₀ of ceftriaxone was shifted to 0.25 μg/mL in the presence of 5ng/mL P1A when added simultaneously and to 128 μg/mL when 50 ng/mL P1Awas preincubated with the antibiotic. Based on this analysis, theconcentrations of P1A and ceftriaxone were adjusted to bracket theinhibitory concentrations of both.

Using concentration of P1A from 5 μg/mL to 50 ng/mL with 0.13 μg/mL to128 μg/mL of ampicillin, the EC₅₀ of ampicillin was shifted from 16μg/mL in the absence of P1A to 128 μg/mL in the presence of 50 μg/mLwhen pre-incubated or added simultaneously. Lower concentrations of P1A(5 μg/mL) had no effect on the ampicillin EC₅₀. The EC₅₀ of ceftriaxonewas shifted from 0.03 μg/mL in the absence of P1A to greater than 4μg/mL in the presence of 50 ng/mL or greater P1A when preincubated oradded simultaneously (Table 13). Lower concentrations of P1A (50 μg/mLto 5 ng/mL) had no effect on the ceftriaxone EC₅₀. Results with thesimultaneous addition of P1A with ampicillin or ceftriaxone wereconfirmed through repeat analysis (Table 14).

TABLE 12 Effective inhibitory concentration (EC₅₀) of ampicillin andceftriaxone in the absence and presence of P1A determined by micro brothdilution analysis. Ampicillin EC₅₀(μg/mL) Ceftriaxone EC₅₀(μg/mL) P1APre Pre Concentration incubation Simultaneous incubation Simultaneous 016 16 <0.13 <0.13 0.5 ng/mL 64 >128 <0.13 <0.13 5 ng/mL 128 >128 <0.130.25 50 ng/mL >128 >128 >128 >128 500 ng/mL >128 >128 >128 >128 5μg/mL >128 >128 >128 >128

Antibiotics were either added to the P1A and incubated for 30 min at 37°C. (Pre-incubation) prior to addition of bacteria, or added to theplates following the addition of P1A and bacteria (Simultaneous). Allconcentrations and combinations were evaluated in triplicate in a96-well plate format. Following overnight incubation, the OD₆₂₅ of theplates was measured in a Spectramax 384 plate reader and the data wereimported into a customized Excel spreadsheet for the determination ofthe 50% effective inhibitory concentration at (EC₅₀) by linearregression analysis. For the purposes of these studies, the statedeffective concentration is the initial concentration of ampicillin orceftriaxone added at the beginning of the experiment, and not aconcentration measured during the course of the experiment or at the endof the incubation period.

TABLE 13 Effective inhibitory concentration (EC₅₀) of ampicillin andceftriaxone in the absence and presence of P1A determined by micro brothdilution analysis. Ampicillin EC₅₀(μg/mL) Ceftriaxone EC₅₀(μg/mL) P1APre Pre Concentration incubation Simultaneous incubation Simultaneous 016 16 0.02 0.02 5 pg/mL 16 16 NA NA 50 pg/mL 128 128 0.02 0.02 500pg/mL >128 >128 0.03 0.03 5 ng/mL >128 >128 0.03 0.03 50ng/mL >128 >128 >4 >4 500 ng/mL NA NA >4 >4

Antibiotics were either added to the P1A and incubated for 30 min at 37°C. (Pre-incubation) prior to addition of bacteria, or added to theplates following the addition of P1A and bacteria (Simultaneous). Allconcentrations and combinations were evaluated in triplicate in a96-well plate format. Following overnight incubation, the OD₆₂₅ of theplates was measured in a Spectramax 384 plate reader and the data wereimported into a customized Excel spreadsheet for the determination ofthe 50% effective inhibitory concentration (EC₅₀).

TABLE 14 Effective inhibitory concentration (EC₅₀) of ampicillin andceftriaxone in the absence and presence of P1A determined by microbrothdilution analysis. P1A Ampicillin EC₅₀(μg/mL) Ceftriaxone EC₅₀(μg/mL)Concentration Simultaneous Simultaneous 0 16 0.03 5 pg/mL 16 NA 50 pg/mL128 0.03 500 pg/mL >128 0.03 5 ng/mL >128 0.03 50 ng/mL >128 >4 500ng/mL NA >4

Antibiotics were added to the P1A bacteria (Simultaneous). Allconcentrations and combinations were evaluated in triplicate in a96-well plate format. Following overnight incubation, the OD₆₂₅ of theplates was measured in a Spectramax 384 plate reader and the data wereimported into a customized Excel spreadsheet for the determination ofthe 50% effective inhibitory concentration at (EC₅₀) by linearregression analysis.

These results demonstrated that P1A effectively inhibited the activityof ampicillin and ceftriaxone in an in vitro microbiological assay andindicated that P1A is 1,000-fold more active against ampicillin thanceftriaxone in the microbiological assay.

Next, the 90% effective inhibitory concentration (EC₉₀) of ampicillin,ceftriaxone, ceftazidime, meropenem, cefepime, cefazolin, amp/sulbactam,cefoperazone, cefotaxime, and cefuroxime against Escherichia coli wasdetermined in the absence and presence of P1A, P3A, or P4A β-lactamase.For the purposes of these studies, the stated effective concentrationwas the initial concentration of ampicillin, ceftriaxone, ceftazidime,meropenem, cefepime, cefazolin, amp/sulbactam, cefoperazone, cefotaxime,and cefuroxime added at the beginning of the experiment, and not aconcentration measured during the course of the experiment or at the endof the incubation period. The results of the analysis are presented inTables 15-17. Each box in the below tables (Tables 15-17) has twonumbers separated by a slash—the two numbers are duplicate assays, allof which showed tight agreement.

Specifically, the assay was performed by diluting (2-fold steps) thechosen antibiotic across the rows of a 96 well dish. The MIC wasdetermined as the point at which the diluted antibiotic transitions fromkilling to not killing the bacteria (in this case, an E. coli strainthat is sensitive to all antibiotics). P1A, P3A, and P4A were diluted(10 fold steps) down the columns (the β-lactamase concentrationsincreased down the columns). As the β-lactamase concentration increased,it reached a level at which it degraded the antibiotic sufficiently toalter the apparent MIC reading (the MIC didn't actually change, but itappeared to change because the amount of antibiotic remaining in thewell was less that the amount loaded into the well). In most cases,there were two break points, the first in which the MIC appeared toincrease a bit, and then at ten-fold concentration higher where the MICexceeded the maximum amount of antibiotic tested. The latter point wasused as the cut-off point. It is important to note that with this E.coli, the MIC was different for each antibiotic. However, for thisassay, the absolute MIC is not the most important parameter. The mostrelevant readout was the β-lactamase concentration at which the apparentMIC increases to the maximum amount of antibiotic tested.

When the cut-off point occurred at low β-lactamase concentration, thisimplied that the β-lactamase was potent at degrading that antibiotic.When it took a lot of β-lactamase to move the MIC, then the β-lactamasewas relatively weak for that antibiotic. This assay was designed,without wishing to be bound by theory to provide predictions of in vivoefficacy for each β-lactamase for a given antibiotic. These data can becombined with the in vitro kinetic data.

TABLE 15 Antibiotic activity of ampicillin and ceftriaxone in thepresence of bacterial culture supernatants from bacillus strainsexpressing soluble β-lactamase proteins P1A (RS310), P3A (IS118), or P4A(IS288). Ampicillin EC₉₀(μg/mL) Ceftriaxone EC₉₀(μg/mL) Rep 1 Rep 2 Rep1 Rep 2 RS310 Concentration 0 2 4 0.008 0.02 5 pg/mL 4 16 NA NA 50 pg/mL32 128 0.008 0.008 500 pg/mL >128 >128 0.008 0.008 5 ng/mL >128 >1280.06 0.008 50 ng/mL >128 >128 >4 >4 500 ng/mL NA NA >4 >4 IS118Concentration 0 2 4 0.008 0.02 5 pg/mL 4 8 NA NA 50 pg/mL 16 64 0.0080.008 500 pg/mL >128 >128 0.5 0.06 5 ng/mL >128 >128 NA >4 50ng/mL >128 >128 >4 >4 500 ng/mL NA NA >4 >4 IS288 Concentration 0 2 40.02 0.02 5 pg/mL 2 16 NA 0.03 50 pg/mL 4 32 0.5 0.13 500 pg/mL 64128 >4 4 5 ng/mL >128 >128 >4 >4 50 ng/mL >128 >128 >4 >4 500 ng/mL NANA >4 NA

E coli 29522 was incubated overnight in the presence of serial dilutionsof antibiotic and the indicated amounts of β-lactamase protein from cellculture supernatants in a 96 well plate. Bacterial density (OD₆₂₅) ofthe cultures was measured and the concentration at which the density wasreduced by 90% relative to an untreated bacterial control (EC₉₀) wasdetermined. The assay was performed on two separate days and the datareported as Rep1 and Rep2. NA=not assayed.

TABLE 16 Antibiotic activity of ceftazidime, meropenem, cefepime, andceftriazone in the presence of bacterial culture supernatants frombacillus strains expressing soluble β-lactamase proteins P1A (RS310),P3A (IS118), or P4A (IS288). Ceftazidime Meropenem Cefepime CeftriazoneEC₉₀ (μg/mL) EC₉₀ (μg/mL) EC₉₀ (μg/mL) EC₉₀ (μg/mL) (high test (hightest (high test (high test 4 μg/mL) 1 μg/mL) 2 μg/mL) 1 μg/mL) RS310Concentration 0 0.13/0.5  0.06/0.06 0.06/0.03 0.06/0.06 50 pg/mL0.25/0.25 0.06/0.03 0.03/0.03 0.06/0.06 500 pg/mL 0.13/0.25 0.06/0.060.06/0.06 0.06/0.13 5 ng/mL 0.13/0.25 0.06/0.06 0.13/0.25 0.25/1.0  50ng/mL 0.5/0.5 0.03/0.06 >2.0/>2.0 >1.0/>1.0 500 ng/mL >4.0/>4.00.03/0.03 >2.0/>2.0 >1.0/>1.0 IS118 Concentration 0 0.13/0.13 0.06/0.030.06/0.03 0.06/0.06 5 pg/mL 0.13/0.25 0.06/0.03 0.03/0.03 0.06/0.13 50pg/mL 0.25/0.25 0.06/0.03 0.06/0.03  1.0/0.25 5 ng/mL 0.13/0.250.06/0.06 0.13/0.13 >1.0/>1.0 50 ng/mL 1.0/2.00.03/0.06 >2.0/>2.0 >1.0/>1.0 500 ng/mL >4.0/>4.00.06/0.06 >2.0/>2.0 >1.0/>1.0 IS288 Concentration 0 0.13/0.5  0.06/0.060.03/0.03 0.06/0.06 50 pg/mL 0.25/0.25 0.06/0.03 0.03/0.03 0.13/>1.0 500pg/mL 0.13/0.25 0.06/0.03 0.06/0.06 >1.0/>1.0 5 ng/mL 0.25/0.5 0.03/0.03 0.25/0.25 >1.0/>1.0 50 ng/mL >4.0/>4.00.03/0.06 >2.0/>2.0 >1.0/>1.0 500 ng/mL >4.0/>4.0 0.06/0.06 >2.0/>2.0>1.0/>1.0

E coli 29522 was incubated overnight in the presence of serial dilutionsof antibiotic and the indicated amounts of β-lactamase protein from cellculture supernatants in a 96 well plate. Bacterial density (OD₆₂₅) ofthe cultures was measured and the concentration at which the density wasreduced by 90% relative to an untreated bacterial control (EC90) wasdetermined. The assay was performed on two separate days and the datareported as two numeric values separated by a slash. NA=not assayed.

TABLE 17 Antibiotic activity of cefazolin, amp/sublactam, cefoperazone,cefotaxime, cefuroxime, and ceftriaxone in the presence of bacterialculture supernatants from bacillus strains expressing solubleβ-lactamase proteins P1A (RS310), P3A (IS118), or P4A (IS288). CefazolinAmp:. Cefuroxime EC₉₀ Sublactam Cefotaxime EC₉₀ Ceftriaxone (μg/mL) EC₉₀(μg/mL) Cefoperazone EC90 (μg/mL) EC₉₀ (high test (high test EC₉₀(μg/mL) (μg/mL) (high test (μg/mL) 64 μg/mL) 128:64 μg/mL) (high test 8μg/mL) (high test 2 μg/mL) 128 μg/mL) (high test 1 μg/mL) RS310Concentration  0 2.0/2.0 4.0:2.0/4.0:2.0 0.25/0.13 0.004/0.008 4.0/4.00.03/0.03  50 pg/mL 2.0/4.0 4.0:2.0/8.0:4.0  0.5/0.25 0.008/0.0044.0/2.0 0.03/0.06 500 pg/mL 16/16 4.0:2.0/4.0:2.0 2.0/4.0 0.004/0.0044.0/4.0 0.13/0.13  5 ng/mL >64/>64 8.0:4.0/16:8.0  >8.01>8.0 0.008/0.00832/32 0.13/0.13  50 ng/mL >64/>64 32:16/32:16 >8.01>8.0 1.0/0.25 >128/>128 >1.01>1.0 500 ng/mL >64/>64128:64/128/64 >8.01>8.0 >2.01>2.0 >128/>128 >1.01>1.0 IS118Concentration  0 2.0/2.0 4.0:2.0/4.0:2.0 0.25/0.25 0.004/0.004 2.0/4.00.03/0.03  50 pg/mL 4.0/4.0 4.0:2.0/8.0:4.0 0.5/0.5 0.004/0.004 8.0/4.00.13/0.13 500 pg/mL 64/64 4.0:2.0/4.0:2.0  8.0/>8.0 0.008/0.008 8.0/8.00.25/0.5   5 ng/mL >64/>64 8.0:4.0/16:8.0  >8.01>8.0 0.06/0.3 128/128 >1.01>1.0  50 ng/mL >64/>6432:16/32:16 >8.01>8.0 >2.01>2.0 >128/>128 >1.01>1.0 500 ng/mL >64/>64128:64/128:64 >8.01>8.0 >2.01>2.0 >128/>128 >1.01>1.0 IS288Concentration  0 2.0/2.0 4.0:2.0/4.0:2.0 0.25/0.13 0.004/0.004 4.0/2.00.03/0.06  50 pg/mL 4.0/4.0 4.0:2.0/4.0:4.0 0.5/0.5  0.02/0.008 8.0/8.00.25/0.25 500 pg/mL >64/>64 4.0:2.0/4.0:2.0 >8.01>8.0 0.25/0.2532/32 >1.01>1.0  5 ng/mL >64/>644.0:2.0/8.0:4.0 >8.01>8.0 >2.01>2.0 >128/>128 >1.0/>1.0  50ng/mL >64/>64 8.0:4.0/8.0:4.0 >8.0/>8.0 >2.0/>2.0 >128/>128 >1.01>1.0500 ng/mL >64/>64 32:16/32:16 >8.0/>8.0 >2.0/>2.0 >128/>128 >1.0/>1.0

E coli 29522 was incubated overnight in the presence of serial dilutionsof antibiotic and the indicated amounts of β-lactamase protein from cellculture supernatants in a 96 well plate. Bacterial density (OD625) ofthe cultures was measured and the concentration at which the density wasreduced by 90% relative to an untreated bacterial control (EC90) wasdetermined. The assay was performed on two separate days and the datareported as two numeric values separated by a slash. NA=not assayed.

Without wishing to be bound by theory, these data suggest more activityin vitro than predicted from the corresponding kinetic data. Forexample, the ceftazidime kinetic data predicted a lack of therapeuticefficacy, but these data showed activity at 500 ng/ml with a 10-foldimprovement for P4A. Next, it was observed that P1A and P3A were quiteeffective for ampicillin, while P4A lost some activity for ampicillin.The amp/sulbactam data verified that sulbactam effectively inhibitedP1A, P3A, and P4A. Whereas the amp/sulbactam data verified thatsulbactam is an effective β-lactamase inhibitor, this should not beinterpreted to mean that these beta-lactamases will not be efficaciousin vivo. Specifically, if the pharamokinetics for biliary excretion aredifferent for the antibiotic and the inhibitor, then the antibiotic canbe found in the small intenstine in the absence of its inhibitor. Forexample, P1A has been shown to be efficacious in humans withpiperacillin/tazobactam. The meropenem data confirmed what was expected,that the P1A, P3A, and P4A β-lactamases are penicillinases andcephalosporinases. Finally, P4A was observed to be remarkably effectiveat degrading ceftriaxone, better than P3A. P4A was as good forceftriaxone as it was for ampicillin. P4 also had improved activity forseveral important cephalosporins with the exception of cefepime. Incomparison to P1A, P4A exhibited about 10-fold or greater 8-lactamaseactivity for ceftriaxone, cefotaxime, ceftazidime, cefazolin,cefoperazone, and cefurxine.

Example 5: Analysis of the Antibiotic Degradation Properties of P1A,P3A, and P4A

P1A and P4A were screened for degradation of ampicillin, ceftriazone,cefotaxime, cefozolin, cefuroxime, cefoperazone, cefepime, andceftazidime in a microtiter plate activity assay that was designed tomimic the activity of the beta-lactamases in the gut in the presence ofhigh antibiotic concentrations. The assay was performed by mixing 10,100 or 1000 μg/ml of the indicated antibiotics with P1A or P4A, atconcentrations of 10 or 100 ng/ml. Plates were either incubated for onehour at 37° C. after which E. coli (ATCC 25922) was added, or E. coliwas added immediately after the addition of the beta-lactamase enzymes.The plates were incubated overnight and the bacterial growth quantifiedby measuring the absorbance at 625 nm (OD₆₂₅) in a Spectramax 384 Plusplate reader. The analysis was performed twice for each beta-lactamaseand antibiotic. The beta-lactamase activity of the culture supernatantswas determined as positive or negative based on the appearance ofbacterial growth in the individual wells. An OD₆₂₅ of 1.0 or greaterindicated maximal bacterial growth, therefore complete antibioticdegradation and high beta-lactamase activity. An OD₆₂₅ of less than 1.0indicated low bacterial growth therefore incomplete antibioticdegradation, hence low beta-lactamase activity.

Results from the analysis are shown in FIG. 8. There was no differencein the readings obtained for the plates that were preincubated with thebeta-lactamase and antibiotic compared to the plates that did notundergo preincubation.

For all antibiotics, except ampicillin, P4A showed imporved activityrelative to P1A. P1A showed no activity for ceftriaxone, cefotaxime, andceftazidime. P4A shows greater efficacy for cefotaxime, cefozolin,cefoperazone, cefepime, and ceftazidime.

Accordingly, modification of five (P4A) amino acids in P1A boostedcephalosporinase activity 10-1000-fold. Oral administration of P4A cantherefore protect the gut flora from commonly used cephalosporins andextend the clinical utility of this prophylactic strategy to theprevention of, for example, CDI. Specifically, the assays presentedherein show that, at concentrations that should be readily achievable inthe small intestine, P4A can degrade cephalosporins and otherantibiotics and have an improved substrate profile relative to P1A.

Definitions

The following definitions are used in connection with the inventiondisclosed herein. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofskill in the art to which this invention belongs.

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disease ofinterest.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

The amount of compositions described herein needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering therapeutic agents (e.g. inventive β-lactamases and/orpharmaceutical compositions (and/or additional agents) for therapeuticpurposes, the therapeutic agents are given at a pharmacologicallyeffective dose. A “pharmacologically effective amount,”“pharmacologically effective dose,” “therapeutically effective amount,”or “effective amount” refers to an amount sufficient to produce thedesired physiological effect or amount capable of achieving the desiredresult, particularly for treating the disorder or disease. An effectiveamount as used herein would include an amount sufficient to, forexample, delay the development of a symptom of the disorder or disease,alter the course of a symptom of the disorder or disease (e.g., slow theprogression of a symptom of the disease), reduce or eliminate one ormore symptoms or manifestations of the disorder or disease, and reversea symptom of a disorder or disease. For example, administration oftherapeutic agents to a patient suffering from a GI tract disorder (e.g.ODD provides a therapeutic benefit not only when the underlyingcondition is eradicated or ameliorated, but also when the patientreports a decrease in the severity or duration of the symptomsassociated with the disease. Therapeutic benefit also includes haltingor slowing the progression of the underlying disease or disorder,regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to about 50% ofthe population) and the ED50 (the dose therapeutically effective inabout 50% of the population). The dosage can vary depending upon thedosage form employed and the route of administration utilized. The doseratio between toxic and therapeutic effects is the therapeutic index andcan be expressed as the ratio LD50/ED50. In some embodiments,compositions and methods that exhibit large therapeutic indices arepreferred. A therapeutically effective dose can be estimated initiallyfrom in vitro assays, including, for example, cell culture assays. Also,a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the 1050 as determined in cellculture, or in an appropriate animal model. Levels of the describedcompositions in plasma can be measured, for example, by high performanceliquid chromatography. The effects of any particular dosage can bemonitored by a suitable bioassay. The dosage can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment.

In certain embodiments, the effect will result in a quantifiable changeof at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 70%, or at least about 90%. In someembodiments, the effect will result in a quantifiable change of about10%, about 20%, about 30%, about 50%, about 70%, or even about 90% ormore. In certain embodiments, the effect will result in a quantifiablechange of two-fold, or three-fold, or four-fold, or five-fold, orten-fold. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder or reduction intoxicity, regardless of whether improvement is realized.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein set forth and as follows in the scope of theappended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

What is claimed is:
 1. A beta-lactamase comprising an amino acidsequence having at least 95% sequence identity with SEQ ID NO: 1 and thefollowing mutations according to Ambler classification: an aliphatichydrophobic residue other than glutamine (Q) at position
 135. 2. Thebeta-lactamase of claim 1, wherein the aliphatic hydrophobic residue isselected from glycine (G), leucine (L), isoleucine (I), methionine (M),and valine (V).
 3. The beta-lactamase of claim 2, wherein the aliphatichydrophobic residue is methionine (M).
 4. A polynucleotide comprising apolynucleotide sequence encoding the beta-lactamase of claim
 1. 5. Ahost cell comprising the polynucleotide of claim
 4. 6. A pharmaceuticalcomposition, comprising the beta-lactamase of claim 1 and apharmaceutically acceptable carrier or excipient.
 7. The pharmaceuticalcomposition of claim 6, wherein the pharmaceutical composition isformulated for oral administration, optionally selected from a tablet,multi-particulate sprinkle, and a multi-particulate capsule.
 8. A methodfor preventing an antibiotic-induced adverse effect in thegastrointestinal (GI) tract, comprising administering an effectiveamount of a beta-lactamase to a patient in need thereof, wherein thebeta-lactamase comprises an amino acid sequence having at least 95%sequence identity with SEQ ID NO: 1 and the following mutationsaccording to Ambler classification: an aliphatic hydrophobic residueother than glutamine (Q) at position
 135. 9. The method of claim 8,wherein the aliphatic hydrophobic residue is selected from glycine (G),leucine (L), isoleucine (I), methionine (M), and valine (V).
 10. Themethod of claim 9, wherein the aliphatic hydrophobic residue ismethionine (M).
 11. The method of claim 8, wherein the subject is beingadministered or will be administered an antibiotic.
 12. The method ofclaim 8, wherein the antibiotic-induced adverse effect in thegastrointestinal (GI) tract is selected from C. difficile infection(CDI), C. difficile-associated disease, and antibiotic-associateddiarrhea (AAD).